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PubMed1974

Studies of the human lymphocyte receptor for heat-aggregated or antigen-complexed immunoglobulin.

The lymphocyte receptor for complexed immunoglobulin was shown not to bind heat-aggregated human serum albumin, bovine serum albumin, transferrin, F(ab')(2), reduced and alkylated Ig, and mildly oxidized Ig, which indicated that the receptor is specific for a site dependent on disulfide bond(s) on the Fc portion of complexed Ig. Inhibition experiments provided evidence that the same receptor binds both heat-aggregated Ig and antigen-antibody complexes. Lymphocytes treated with pronase were no longer able to bind Ig complexes, which suggested that the receptor is a protein or glycoprotein. Additional evidence was obtained that lymphocyte surface Ig and the receptor for complexed Ig are distinct since the former could be capped without affecting the distribution of the latter, and surface Ig was not detectable after trypsinization of lymphocytes, whereas the binding of Ig complexes was unaffected by such treatment. Incubation of lymphocytes which had bound Ig complexes in tissue culture medium at 37 degrees C revealed that the complexes remained on the surface membrane for several hours, and that only a minority of lymphocytes binding complexes showed cap formation. Lymphocytes which had heat-aggregated IgG specifically bound to their receptors for complexed Ig were markedly inhibited in their ability to mediate antibody-dependent cytotoxicity, thus providing strong evidence for the necessity of the receptor in this immune activity. Titration of this inhibition with varying amounts of complexes revealed distinct plateaus in the dose-response curve. This suggested that there may be more than one kind of receptor and/or different populations of lymphocytes which bear the receptor.

Animals↗
PubMed1994

Surfactant protein A amino acids Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells.

Pulmonary surfactant protein A (SP-A) is a mammalian lectin that regulates the uptake and secretion of surfactant by alveolar type II cells and is an important component of surfactant complexes. The domains of SP-A which mediate these functions have not been fully mapped. The binding of SP-A to its high affinity receptor on alveolar type II cells is thought to be dependent on a carbohydrate recognition domain (CRD), while the interaction with lipids has been attributed to the hydrophobic neck region of the molecule. To explore the role of the CRD in the interactions of SP-A with type II cells and lipids, we introduced mutations into the cDNA to encode for the substitutions Glu195-->Gln and Arg197-->Asp (SP-Ahyp,Gln195,Asp197) and expressed the mutant protein in insect (Sf9) cells using recombinant baculoviruses. Similar mutations introduced into mannose-binding protein A have been shown to switch the carbohydrate binding specificity from mannose > galactose to the converse. Wild type SP-A produced in Sf9 cells does not contain hydroxyproline (SP-Ahyp), but like rat SP-A it binds to carbohydrate affinity columns, lipids, and the SP-A receptor and is a potent inhibitor of the secretion of surfactant from type II cells (IC50 = 0.5-1.0 micrograms/ml). The SP-Ahyp,Gln195,Asp197 also bound to affinity matrices of galactose-Sepharose and mannose-Sepharose but the indicated mutations rendered the binding at least 100 times more susceptible than SP-Ahyp to competition by free galactose. The SP-Ahyp,Gln195,Asp197 did not compete with rat SP-A for occupancy of its high affinity receptor on type II cells and the mutant protein was 25-50-fold less potent as an inhibitor of the secretion of surfactant from type II cells (IC50 = 26.0 micrograms/ml). Unlike SP-Ahyp, the inhibition of secretion of surfactant by SP-Ahyp,Gln195,Asp197 was reversed by 0.25 M alpha-methylmannoside or galactose. In addition, the SP-Ahyp,Gln195,Asp197 bound avidly to phospholipid but did not aggregate vesicles or augment the uptake of phospholipid into type II cells. We conclude that the binding of SP-A to its receptor and the inhibition of surfactant secretion are critically dependent on the carbohydrate binding specificity of the CRD. Furthermore, phospholipid aggregation and augmentation of phospholipid uptake into type II cells are mediated by the COOH-terminal region of SP-A by a mechanism that is distinct from phospholipid binding.

Animals↗
PubMed1988

High affinity specific binding sites for tritiated platelet-activating factor in canine platelet membranes: counterparts of platelet-activating factor receptors mediating platelet aggregation.

In canine platelet membranes, tritiated platelet activating factor (PAF) labels in a saturable and reversible manner a single population (nH = 0.97) of binding sites. The affinity of this binding was high (Kd = 0.23 +/- 0.02 nM, n = 4, and 0.21 +/- 0.05, n = 8, determined by kinetics or saturation experiments, respectively), and the density of binding sites (Bmax) was 911 +/- 31 fmol/mg of protein (n = 8). [3H]PAF binding was entirely reversed by unlabeled PAF (10 microM). [3H]PAF exhibited stereoselective discrimination inasmuch as it was poorly displaced by enantio-PAF, the PAF enantiomer that does not occur naturally. Furthermore, the displacing potency of the (+)-enantiomer of the PAF antagonist 52770 RP against [3H]PAF was 45 times higher than that of the (-)-enantiomer. [3H]PAF binding displayed a remarkable specificity in that it was not affected by a variety of classical pharmacological agents. However, this binding was displaced by several PAF receptor antagonists such as 59227 RP, CV-6209, Ro 19-3704, 52770 RP, brotizolam, WEB 2086, SRI 63-441, L-652,731, alprazolam, triazolam, and BN 52021. The Ki of the 16 studied antagonists ranged from 7.9 nM (59227 RP, most potent) to 16.8 microM (BN 52021, least potent). The possible biological significance of our binding procedure was assessed by correlating the potencies of 16 PAF antagonists as [3H]PAF displacers in dog platelet membranes and as inhibitors of PAF-induced platelet aggregation in washed canine platelets. This analysis revealed the existence of a highly significant correlation (r = 0.82, p less than 0.001) between biochemical and functional tests. However, two compounds (Ro 19-3704 and BN 52021) were found to be located outside the confidence limits when the probability level of belonging to the regression line was set at 0.01. In conclusion, this study provides evidence that [3H]PAF binding in canine platelet membranes exhibits the required properties for a valid binding procedure. Furthermore, the labeled sites are likely to be the counterparts of platelet receptors that, when activated by PAF, induce aggregation.

Animals↗
PubMed2004

Autonomous SHIP-dependent FcgammaR signaling in pre-B cells leads to inhibition of cell migration and induction of cell death.

Mature B cells express a single immunoglobulin Fc receptor, FcgammaRIIB, that functions to block downstream signaling by co-aggregated antigen receptors. Co-aggregation of receptors is essential because BCR activated kinases must phosphorylate FcgammaRIIB to recruit SHIP and mediate inhibitory signals. Pre-B cells also express FcgammaRIIB, but since they do not yet express antigen receptor, it is unclear when they are activated physiologically. Here, we demonstrate that aggregation of the FcR on pre-B cells leads to potent inhibitory signaling. Aggregation of the FcR alone leads to downstream effects including the induction of cell death and the blockade of SDF-1 induced migration. The biochemical circuitry that mediates this response is unique because although SHIP is required for this signaling and is phosphorylated upon receptor aggregation, this occurs in the absence of FcgammaRIIB phosphorylation. Results indicate that immune complexes may inhibit B cell production in the bone marrow by antigen non-specific mechanisms.

Animals↗
PubMed1996

NMDA receptors play an anti-aggregating role in human platelets.

Receptors for different monoamines, peptides and other neurohormones are present in the plasma membrane of platelets, and the sophisticated process of haemostasis is regulated by the interplay of their physiologic agonists (1). The recent report of a platelet binding site for phencyclidine (2) suggested a possible role of N-methyl-D-aspartate (NMDA) receptors in platelet function. Isotherms of [3H]-glutamate (GLU), [3H]-CGP-39653, [3H]-glycine (GLY) and [3H]-MK-801 carried out in platelet membranes yielded Bmax and Kd values for these ligands similar to those present in neurons, and NMDA only partially displaced [3H]-GLU. In neurons [3H]-MK-801 binding is potentiated by GLU and/or GLY and, being specific for the open NMDA receptor channel species, it has a functional meaning. In platelet membranes neither GLU and/or GLY increased [3H]-MK-801 binding; thus suggesting that NMDA receptors in platelets are different from those present in neurons. GLU or NMDA alone did not induce platelet aggregation. However, both amino acids were antagonistic on the aggregating activity of arachidonic acid (AA), NMDA being 3 orders of magnitude more active than GLU, and NMDA also antagonized adenosine diphosphate (ADP) and platelet aggregating factor (PAF) induced platelet aggregation. Finally, NMDA increased cAMP levels in intact platelets, and such an effect did not occur in a Ca(2+)-free medium; yet, cAMP increase was not antagonized by the calmodulin inhibitor trifluoperazine (TFP). It was concluded that platelet membranes carry an NMDA receptor, functionally distinct from the neuronal one, which seems to play an anti-aggregating role.

Cyclic AMP↗
PubMed2003

Effects of roxifiban on platelet aggregation and major receptor expression in patients with coronary artery disease for the Roxifiban Oral Compound Kinetics Evaluation Trial-I (ROCKET-I Platelet Substudy).

BACKGROUND: It has been expected that therapy with oral glycoprotein (GP) IIb/IIIa blockers including roxifiban will reduce mortality and vascular complications in a long-term. However, platelet-related properties of roxifiban in the clinical setting are not well known. We measured platelet characteristics during chronic treatment in patients with coronary artery disease enrolled in the Roxifiban Oral Compound Kinetics Evaluation Trial (ROCKET-I). METHODS: ROCKET-I was designed as a randomized, double blind, multicenter, dose-ranging study of roxifiban, administered either as monotherapy or concomitantly with aspirin, compared with aspirin alone. Thirty-one patients were assigned for 24 weeks of therapy with aspirin (n = 7), roxifiban (n = 9), or roxifiban plus aspirin (n = 15). Platelets were assessed 5 times in each patient at baseline, and at weeks 2, 4, 12, 18, and 24 thereafter with aggregometry and flow cytometry. RESULTS: Baseline platelet characteristics were similar in all 3 groups. There was a consistent significant decrease of adenosine diphosphate- (P =.0001) and collagen-induced (P =.002) platelet aggregation in the patients treated with roxifiban when compared with patients treated with aspirin alone. Flow cytometry revealed paradoxical late activation of GP IIb/IIIa expression (P =.007) when roxifiban was used without aspirin, which was significant compared with the aspirin and aspirin-roxifiban groups. There were no differences among groups in GP Ib expression, although its rise was more profound in the patients treated with roxifiban. There were substantial differences in the P-selectin expression. Although aspirin time dependently decreased the percent of P-selectin positive platelets (P =.02), treatment with roxifiban resulted in the phasic changes with the early inhibition (P =.01) and then 2-fold activation (P =.0001) starting at week 12 of the therapy. There was an early transient activation of platelet endothelial cell adhesion molecule-1 expression (P =.008) at week 2, followed by the later inhibition of this receptor (P =.003) in patients treated with roxifiban. CONCLUSION: Despite achieving sustained inhibition of platelet aggregation, therapy with roxifiban has been associated with over expression or phasic changes of major platelet receptors. These data may explain clinical concerns about the use of oral GP IIb/IIIa inhibitors linking higher mortality rates and incidence of thrombotic episodes with paradoxical switching to alternative passways of platelet activation.

Amidines↗
PubMed2003

Effects of clopidogrel and aspirin combination versus aspirin alone on platelet aggregation and major receptor expression in patients with heart failure: the Plavix Use for Treatment Of Congestive Heart Failure (PLUTO-CHF) trial.

BACKGROUND: Persistent platelet activation may contribute to thrombotic events in patients with congestive heart failure (CHF). Chronic use of mild platelet inhibitors could therefore represent an independent avenue to improve morbidity, mortality, and quality of life in this expanding population. Although clopidogrel is widely used in patients with acute coronary syndromes and ischemic stroke, the ability of this novel ADP-receptor antagonist to inhibit platelet function in patients with CHF is unknown. We assessed antiplatelet properties of clopidogrel with aspirin (C+A) versus aspirin alone (A) in patients with CHF with heightened platelet activity. METHODS: Patients with left ventricular ejection fraction <40%, or CHF symptoms in the setting of preserved systolic function and New York Heart Association class II-IV were screened. Patients were considered to have platelet activation when 4 of the following 5 parameters were met: ADP-induced platelet aggregation >60%; collagen-induced aggregation >70%; whole blood aggregation >18 ohms; expression of GP IIb/IIIa >220 log MFI; and P-selectin cell positivity >8%. All patients were treated with 325 mg of acetylsalycilic acid (ASA) for at least 1 month. Patients receiving an antithrombotic agent other than ASA were excluded. Patients meeting clinical and laboratory criteria were randomly assigned to C+A (n=25), A (n=25) groups, or represent screen failures (n=38). Platelet studies (conventional and whole blood aggregometry, shear-induced activation, expression of 10 major receptors and formation of platelet-leukocyte microparticles) were performed at baseline and after 30 days of therapy. RESULTS: There were no deaths, hospitalizations, or serious adverse events. There were no changes in platelet parameters in the A group. In contrast, therapy with C+A resulted in a significant inhibition of platelet activity assessed by ADP-induced (P =.00001), and epinephrine-induced (P =.0016) aggregation, closure time (P =.04), expression of PECAM-1 (P =.009), GP Ib (P =.006), GP IIb/IIIa antigen (P =.0001), GP IIb/IIIa activity with PAC-1 (P =.0021), and CD151 (P =.0026) when compared with the A group. Therapy with C+A also resulted in the reduced formation of platelet-leukocyte microparticles (P =.021). Collagen-induced aggregation in plasma and in whole blood, expression of vitronectin receptor, P-selectin, CD63, CD107a, and CD107b did not differ among groups. CONCLUSIONS: Treatment with C+A for 1 month provides significantly greater inhibition of platelet activity than ASA alone in patients with CHF. Patients with CHF with heightened platelet activity represent a potential target population in which addition of clopidogrel may decrease mortality rates by reducing the incidence of thrombotic vascular events.

Aged↗
PubMed2000

Baseline platelet aggregation and major receptor expression predict subsequent activity following thrombolysis for acute myocardial infarction.

The early identification of patients with heightened platelet activity for aggressive antiplatelet regimens represents a critical clinical issue in acute myocardial infarction (AMI) therapy. We sought to determine whether the degree of pre-reperfusion platelet function is related to subsequent activity following thrombolysis. Platelets were investigated at baseline and at 24 h following thrombolytic therapy by aggregometry, and flow cytometry in 19 AMI patients enrolled in the GUSTO-III trial. Regression analysis revealed a significant correlation for 5 microM ADP (r2 = 0.529), 10 microM ADP (r2 = 0.445), thrombin (r2 = 0.226), collagen (r2 = 0.568), and ristocetin-induced aggregation (r2 = 0.964). Platelet expression linearly correlated for GPIIb/IIIa (r2 = 0.337), P-selectin (r = 0.817), PECAM-1 (r2 = 0.586), and the vitronectin receptor (r2 = 0.634). The data suggest that the baseline characteristics predict future platelet activity, and may prospectively identify patients who will benefit most from antiplatelet strategies after coronary thrombolysis.

Biomarkers↗
PubMed2001

Activation of Gi-coupled receptors releases a tonic state of inhibited platelet aggregation.

Single-receptor pharmacology does not satisfactorily explain the physiology of the ADP-induced platelet aggregation response. It has been shown that, in addition to Gq-coupled receptor activation, one Gi-coupled receptor, either the ADP P2T or the alpha2-adrenoceptor, is required for elicitation of aggregation. The underlying mechanism of this action, however, has not been elucidated. By systematically assaying the entire time course of the aggregation and its fade using two methods of aggregometry, we have investigated the role of graded activation of these two Gi-coupled receptors. We demonstrate that constant activation of either of two Gq-coupled receptors, the ADP P2Y1 or the 5-HT2A, and incremental activation of either of the two Gi-coupled receptors, tightly regulates the aggregation response in vitro, through the apparent release of a tonic inhibition of platelet aggregation. This tightly regulated release of inhibition, which appears analogous to the phenomena of disinhibition observed in the central nervous system, may be instrumental for the continuous adaptation of the aggregation response to variable physiological conditions.

Adaptation, Physiological↗
PubMed1992

Interaction of aggregated native and mutant IgE receptors with the cellular skeleton.

When aggregated, cell surface proteins become resistant to solubilization by detergents, presumably because of aggregation-induced or -stabilized interactions between the membrane protein and the cytoskeleton or plasma membrane skeleton. We genetically engineered variants of the tetrameric high-affinity receptor for IgE (Fc epsilon RI) to identify a site on its alpha, beta, or gamma chains that mediates such putative interactions. Using flow cytofluorometry, we studied rat basophilic leukemia cells, transiently transfected COS cells, and stably transfected P815 cells bearing wild-type and mutated receptors. We observed that (i) solubilization was markedly dependent on the degree of aggregation, the extent varying somewhat with the cell type and, particularly at lower levels of aggregation, with the time after addition of detergent; (ii) truncation of no single cytoplasmic domain of the alpha, beta, or gamma chains ablated the insolubilization effect; and (iii) incomplete receptors were also efficiently insolubilized by aggregation. Thus receptors consisting only of alpha and gamma chains, a "receptor" consisting of only the ectodomain of the alpha chain attached to the plasma membrane by a glycosyl-phosphatidyl inositol anchor, and "receptors" consisting only of minimally modified gamma chains were resistant to solubilization after aggregation. We conclude that no unique subunit or domain of Fc epsilon RI mediates the insolubilization phenomenon. Our results support a model in which the bridging of membrane proteins leads to their becoming nonspecifically enmeshed in a network of membrane skeletal proteins on either the outside and/or the inside of the membrane so that dissolution of the lipid bilayer becomes irrelevant.

Animals↗
PubMed1983

Direct observation of the rapid aggregation of acetylcholine receptors on identified cultured myotubes after exposure to embryonic brain extract.

We have directly observed the redistribution of acetylcholine receptors (AChR) on the surface of cultured myotubes, induced by a soluble brain extract. The AChR were fluorescently labeled with rhodamine-conjugated alpha-bungarotoxin and viewed under low incident illumination with a video image intensification system. The results of our sequential observations indicate that AChR aggregates can be assembled rapidly (30-120 min) from mobile, diffuse AChR. This assembly was characterized by the initial formation of microaggregates (less than 1 micron diameter) that increased in number and coalesced or grew to form larger aggregates. The redistribution of fluorescently labeled AChR was completely inhibited by illumination of cells at levels used for conventional fluorescence micrography and could be observed only by using low light levels.

Animals↗
PubMed1993

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

Adaptor Protein Complex 2↗
PubMed1978

Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells.

We have studied in detail the binding of fluorescent derivatives of insulin and epidermal growth factor to 3T3 fibroblasts. We have used two types of fluorescent analogues of insulin and epidermal growth factor: highly fluorescent derivatives which have seven to eight rhodamine molecules or fluorescent derivatives which have a single rhodamine molecule per one molecule of insulin or epidermal growth factor. Both types of analogue retained substantial binding affinity as determined by radioreceptor assays and biological activity. The cells labeled with the fluorescent analogues were visualized with a sensitive video intensification microscopic system that enabled us to directly observe the location of the fluorescent hormone on the surface and within the living fibroblasts. We found that both insulin and epidermal growth factor initially bound diffusely to the cell surface and, at 4 degrees , remained dispersed. Within a few minutes at 23 degrees or 37 degrees the hormone-receptor complexes aggregated into patches that could be readily removed by trypsin but not by excess native hormone. The hormone-receptor complexes, which were initially mobile in the plane of the membrane, become immobilized later as the consequence of the receptor aggregation or internalization. Within approximately 30 min at 37 degrees , much of the labeled hormone was found within the cell in endocytic vesicles that moved about in the cytoplasm in a saltatory manner. The aggregation and immobilization of the hormone-receptor complexes could be due to either hormone-hormone interactions on the cell membrane or a hormone-induced conformational change in the hormone-receptor complex. Aggregation and internalization of hormone-receptor complexes could be associated with certain aspects of hormone action, hormone degradation, down regulation of receptors, or negative cooperativity of hormone binding.

Cell Line↗
PubMedDate not supplied

Characterization of the detergent insolubility of the T cell receptor for antigen.

Aggregation of cell surface receptors plays an important role in signal transduction in many receptor systems. In the T cell receptor (TCR), as in many other cell surface receptors, this aggregation results in insolubility in certain nonionic detergents. We have characterized this insolubility for TCR, and we show it is not preexisting in HPB-ALL cells but increases with increasing TCR aggregation. It is not likely to be due to a direct interaction with cellular cytoskeletal elements, as it is not affected by inhibitors of actin or tubulin polymerization. It may be due to interaction with detergent-resistant membrane domains that have been found in various cell types and contain tyrosine kinases, the earliest known participants in TCR signal transduction. This aggregation-dependent insolubility occurs as rapidly as the anti-TCR antibody binds, so the kinetics are consistent with an involvement in signal transduction. It is not, however, dependent on signal transduction, as inhibitors of tyrosine kinases do not inhibit the insolubility. Insolubility is also enhanced by preaggregation of CD4, an important T cell surface molecule which also associates with the tyrosine kinase p56lck.

CD4 Antigens↗
PubMed1992

Does co-aggregation of the CD45 and CD3 antigens inhibit T cell antigen receptor complex-mediated activation of phospholipase C and protein kinase C?

The binding of agonistic monoclonal antibodies (mAb) to the CD3 antigen in T cells induces a rapid increase in tyrosine phosphorylation, inositive phosphate (IP) production, a rise in intracellular calcium and protein kinase C (PKC) activation. These intracellular signals have been implicated in the control of interleukin-2 and interleukin-2R receptor gene expression, thereby regulating T cell proliferation. Previous studies have shown that co-ligation of the CD45 and CD3 antigens inhibits CD3-induced tyrosine phosphorylation, IP production, calcium signals and T cell proliferation. It has therefore been suggested that the CD45 antigen uncouples the T cell receptor (TcR) from mitogenic signal pathways. In this study co-ligation of the CD3 and CD45 antigens with precisely constructed bispecific mAb did not inhibit CD3-induced T cell proliferation, IP production, calcium signals, diacylglycerol production or PKC activation. Furthermore, co-ligation of CD3 and CD45 antigens already cross-linked with IgM mAb did not lead to inhibition of CD3-induced calcium signals. Inhibitions of CD3-induced intracellular signals were observed following co-ligation of IgG CD45 and CD3 mAb with anti-IgG (F(ab')2 fragments. However, comparable inhibitions were also noted following co-ligation of CD3 with other abundant cell-surface antigens such as CD5 and LFA-1, and inhibitions were only observed when the CD3 mAb used required cross-linking to induce signals. These results suggested that the inhibitory effects of CD45 IgG mAb were not specific and were caused by the prevention of CD3-CD3 cross-linking following CD3 antigen co-ligation with other cell surface molecules. These findings are inconsistent with a specific inhibitory role for the CD45 phosphotyrosine phosphatase in uncoupling the TcR from mitogenic signal pathways.

Antibodies, Monoclonal↗
PubMed1988

Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗