The interaction of IgE with rat basophilic leukemia cells. II. Quantitative aspects of the binding reaction.

Previous studies indicated that cultured rat basophilic leukemia cells have surface receptors which bind IgE with high specificity. In this paper we describe some quantitative aspects of the phenomenon. The reaction mechanism appears to consist of a simple reversible binding reaction with k(1) = 9.6 x 10(4) M(-1) sec(-1) and k(-1) 1.6 x 10(-5) sec(-1) at 37 degrees C. The calculated K(A) is therefore 6 x 10(9) M(-1). The activation energy of binding was found to be 7.8 kcal/mol. The number of binding sites/cell varied between 3 x 10(5) to over 1 x 10(6). The binding was insensitive to pH's between 6-8 but at pH 3.0 complete dissociation of bound IgE occurred in approximately 1 min at 0 degrees C leaving the receptors for IgE intact. Ca(++) plus EDTA and Mg(++) plus EDTA produce a fairly marked reduction in binding capacity though these reagents alone produce much smaller effects.

The interaction of IgE with rat basophilic leukemia cells. I. Evidence for specific binding of IgE.

A rat basophilic leukemia cell line originally described by Eccleston et al. (3) has been successfully transplanted into eight Wistar strains and adapted to suspension cell culture without noticeable morphological changes. The cells deplete the reaginic activity of mouse and rat immune sera to an extent roughly equivalent to that reported for normal rat mast cells. Studies utilizing radioiodinated antilight-chain antibodies and radioiodinated partially purified rat IgE indicate that the cells bind IgE to their surface membrane with high specificity. Heating or mildly reducing the rat IgE destroyed its binding activity. The binding is unaffected by the presence or absence of Ca(++) and Mg(++), and is markedly inhibited by reaginic but not by normal rat or mouse serums. The affinity of these cells for human IgE, if present at all, is substantially lower than for rat IgE.

Electron microscopic localization of immunoglobulin E on the surface membrane of human basophils.

We have examined human leukocyte preparations for the presence of surface-bound IgE by electron microscopy. Basophil-enriched leukocytes were reacted with burro anti-IgE, a hybrid antibody to burro IgG and ferritin, and ferritin, with or without prior incubation of the cells with an IgE myeloma protein. In the absence of preincubation with IgE small amounts of ferritin were fixed to the surface of basophils but on no other cells. When cells were preincubated with IgE the amount of ferritin fixation on the basophils was markedly increased and a small amount of ferritin was also bound to platelets but again to no other leukocytes. The distribution of ferritin on the basophil surface appeared dependent upon the temperature at which the cells were kept during and after reaction with the various reagents. Basophil sections from cells kept at 0 degrees C had ferritin bound to the surface membrane in patches distributed around the entire circumference. Basophil sections from cells prepared at room temperature had ferritin distributed assymetrically covering a surface membrane segment one-fifth to one-half of the circumference. In control studies in which monomeric IgG was substituted for the IgE and burro anti-IgG was used instead of burro anti-IgE, no cellular fixation of ferritin was observed.

Cell cycle-associated changes in receptors for IgE during growth and differentiation of a rat basophilic leukemia cell line.

The rat basophilic leukemia cell line (RBL-1) showed an inverse relationship between growth rate and expression of receptor activity for IgE. After prolonged exponential growth, the number of receptors per cell stabilized at 4-6 times 10-5. Cells in stationary cultures, which are arrested in the G1 phase of the cell cycle, continued to accumulate up to 0.9-1.7 times 10-6 receptors/cell with no increase in volume. Upon resuspension in fresh medium at low density, these cells were shown to lose up to 70% of the receptor activity within 4 h. Assessment of cultures synchronized by double thymidine block and cells fractionated by centrifugation of a Ficoll gradient indicated that the RBL-1 cells acquire receptors in the G1 phase of the cell cycle. No accumulation of active receptors occurred during the S and G2 phases, though the average cell volume increased. Cell division resulted in a drop in number of receptors per cell while the number of cell-bound receptors in the culture remained unchanged. This indicates that during mitosis receptors were simply distributed to daughter cells.

Characterization of gene expression in resting and activated mast cells.

To characterize gene expression in activated mast cells more comprehensively than heretofore, we surveyed the changes in genetic transcripts by the method of serial analysis of gene expression in the RBL-2H3 line of rat mast cells before and after they were stimulated through their receptors with high affinity for immunoglobulin E (FcepsilonRI). A total of 40,759 transcripts derived from 11,300 genes were analyzed. Among the diverse genes that had not been previously associated with mast cells and that were constitutively expressed were those for the cytokine macrophage migration inhibitory factor neurohormone receptors such as growth hormone- releasing factor and melatonin and components of the exocytotic machinery. In addition, several dozen transcripts were differentially expressed in response to antigen-induced clustering of the FcepsilonRI. Included among these were the genes for preprorelaxin, mitogen-activated protein kinase kinase 3, and the dual specificity protein phosphatase, rVH6. Significantly, the majority of genes differentially expressed in this well-studied model of mast cell activation have not been identified before this analysis.

Surface IgE on human basophils during histamine release.

The distribution of surface IgE on human basophils was studied using fluorescence microscopy and immunoferritin electronmicroscopy. Redistribution of the IgE was dose, time and temperature dependent and required divalent anti-IgE. Cells which can release histamine in vitro were indistinguishable in these respects from cells which cannot. The redistribution was unaffected by the presence or absence of Ca(++). No correlation between the conditions required for optimal histamine release and for redistribution was observed. At low doses, optimal histamine release occurred in the absence of, or before, redistribution. At higher doses redistribution occurred in the absence of histamine release. Antigen-induced histamine release was unaccompanied by gross redistribution of the surface IgE. Since both histamine release and redistribution require bridging of IgE on basophils it is concluded that only certain kinds of cross-linking of the IgE effectively stimulates these cells.

Affinity site labeling of a mouse myeloma protein which binds dinitrophenyl ligands.

A mouse myeloma protein of the immunoglobulin A class, which specifically binds dinitrophenyl ligands, has been successfully affinity labeled (site-directed labeling) with several diazonium reagents, leading to inactivation of the combining sites. The labeling reagents reacted only with tyrosine residues of light chain origin. The 7S subunit of the myeloma protein appears to contain only one reactive site.

An unusual mechanism for ligand antagonism.

The ratio of late to early events stimulated by the mast cell receptor for immunoglobulin E (IgE) correlated with the affinity of a ligand for the receptor-bound IgE. Because excess receptors clustered by a weakly binding ligand could hoard a critical initiating kinase, they prevented the outnumbered clusters engendered by the high-affinity ligands from launching the more complete cascade. A similar mechanism could explain the antagonistic action of some peptides on the activation of T cells.

Evolution of immunoglobulin polypeptide chains: carboxy-terminal of an IgM heavy chain.

The dipeptide sequence at the carboxy-terminal of a heavy (micro) chain from a human macroglobulin ( IgM) is tyrosylcysteine, although the reverse sequence, cysteinyltyrosine, has not been rigorously excluded. The presence of cysteine at the carboxy-terminal was predicted from a recognition of the chemical homologies among the polypeptide chains of immunoglobulins, and their probable evolutionary origin.

Expression of high-affinity binding of human immunoglobulin E by transfected cells.

The receptor with high affinity for immunoglobulin E (IgE) on mast cells and basophils is critical in initiating allergic reactions. It is composed of an IgE-binding alpha subunit, a beta subunit, and two gamma subunits. The human alpha subunit was expressed on transfected cells in the presence of rat beta and gamma subunits or in the presence of the gamma subunit alone. The IgE binding properties of the expressed human alpha were characteristic of receptors on normal human cells. These results now permit a systematic analysis of human IgE binding and a search for therapeutically useful inhibitors of that binding.

Protein dimerization. Inside job.

In a sophisticated combination of genetic engineering and organic synthesis, a general method for dimerizing recombinant intracellular proteins has been devised; the usefulness of the method should now be testable.

Immunoglobulin receptors. Handicapping the immune response.

Gene targeting experiments that "knock out" the expression of cellular Fc receptors for immune complexes have confirmed previous assumptions about the receptors' function but have also revealed some surprises.

Fc receptors and membrane immunoglobulin.

This year there have been three major highlights in this field: genetic sequencing of the principal Fc receptors has been completed; new proteins associated with both Fc gamma receptors and B-cell membrane immunoglobulins have been identified; and there has been an initial analysis of structural-functional relationships in these receptors using genetic engineering.

Secretion from rat basophilic leukaemia cells induced by calcium ionophores. Effect of pH and metabolic inhibition.

Previous experiments on the functional properties of rat basophilic leukaemia cells showed a major anomaly when compared to normal mast cells: though IgE-mediated secretion was dependent on external Ca2+ with both types of cells, substantial non-cytotoxic release with ionophore A23187 could be demonstrated with the normal cells but not with the tumour cells. We now show that when the pH of the incubation medium is increased to 8 it is possible to obtain excellent Ca-dependent, non-cytotoxic secretion from tumour basophils with the ionophores A23187 and ionomycin. These results provide further evidence that secretion from the tumour cells occurs via a mechanism similar to that used by normal mast cells and basophils. Experiments with metabolically inhibited tumour cells suggest that their unusual sensitivity to the cytotoxic effects of Ca2+ ionophores may be related to their ability to sequester intracellular calcium. Changes in the conditions of cell culture appeared to produce substantial and at least partially reversible changes in responsiveness to IgE-mediated triggering and ionophores.

Energy-dispersive X-ray reflectivity study of the model membranes at the air/water interface.

The results of the first energy-dispersive reflectivity measurements with a (membrane coated) liquid surface are reported. They rely on the calibration curve measured with pure water and can be done without any sample or detector movement with a low-intensity, laboratory-based X-ray generator within less than 1 h. As an illustration, the structural parameters of a diarachidoylphosphatidylcholine monolayer at the air/water interface are determined. It is argued that the energy-dispersive detection in combination with the intense synchrotron radiation can be used for the time-resolved reflectivity measurements on the time-scale of minutes.

Gel filtration in 6 M guanidine hydrochloride of the alpha-subunit (and its fragments) of the receptor for immunoglobulin E.

The mol. wts of the alpha-chain of the receptor for immunoglobulin E and several of its enzyme-cleaved fragments have been evaluated by gel filtration on Sepharose 6B in 6 M guanidine HCl. The mol. wt of alpha-chains treated with endoglycosidase was 30% less than that of untreated alpha-chains. alpha-Chains digested with papain eluted in a single peak with a mol. wt approximately one-half of that of undigested alpha-chains. The results support the proposal that papain cleaves alpha-chains into two fragments of similar size [Goetze et al. (1981) Biochemistry 20, 6341-6349].

The receptor for immunoglobulin E: examination for kinase activity and as a substrate for kinases.

Purified receptor-immunoglobulin E (IgE) complexes incubated with [gamma-32P]-ATP incorporated phosphorus into tyrosines on the beta and gamma chains of the receptor. The activity had the typical characteristics of a tyrosine kinase. Immunoprecipitation of the complexes with anti-IgE left the activity in the supernatant, demonstrating that the receptor itself was not the kinase. The receptor was also phosphorylated when membranes or intact cells were incubated with radioactive ATP or phosphate, respectively, but in each case the subunits or amino acid residues that were modified were distinctive.

Further characterization of the beta-component of the receptor for immunoglobulin E.

A 30,000 mol. wt component (beta) is associated in a 1:1 ratio with the 50,000 mol. wt glycoprotein (alpha) which binds immunoglobulin E (IgE) on mast cells and related tumor cells. We show that alpha and beta are associated in membrane preparations. This is consistent with previous results which showed labeling of beta with the hydrophobic probe 5-iodonaphthyl-1-azide (INA). The beta-polypeptide is susceptible to proteolytic cleavage during preparation of the membranes and when this occurs a 20,000 fragment can be labeled with INA and remains associated with alpha. No incorporation of carbohydrate precursors into beta was observed. Since beta is also not modified when cells are surface-labeled, it may not be exposed on the cell surface. Rigorous washing of IgE-receptor complexes with non-ionic detergent results in dissociation of beta from the alpha-IgE complex. The latter will then not reassociate with beta when exposed to crude detergent extracts of the tumor cells.

Proteolysis of soluble IgE-receptor complexes: localization of sites on IgE which interact with the Fc receptor.

Mouse and rat IgE and the respective soluble IgE-receptor complexes purified from rat basophilic leukemia cells were digested with trypsin. The end product in each case was F(ab')2-like. It contained the Ce2 regions, had intact antigen combining sites but had lost all ability to bind to the cell receptor for IgE. With mouse IgE the two principal sites of cleavage are likely to be the interdomain regions between Ce4:Ce3 and Ce3:C32 respectively. Cleavage at these sites occurs sequentially with the rate constant for the cleavage at the second site being approximately four-fold greater than that for the initial cleavage. When IgE is bound to the receptor the rates of cleavage are inhibited approximately three-fold. With rat IgE, the principal initial cleavage occurs within the intrachain disulfide loop in the Ce3 domain. Even when this disulfide bond in the digested protein is reduced, the product retains a substantial binding activity. A second cleavage occurs at a similar rate as the first and at a site analogous to that seen with mouse IgE, i.e. between the Ce3 and Ce2 domains. Notably, when bound to the receptor, the rate of cleavage at the first site is inhibited approximately three-fold but at the second site more than or equal to 40-fold. These results strongly implicate thd Ce3 domain as the principal site of interaction between rodent IgE and its receptor.

Coordinate synthesis and degradation of the alpha-, beta- and gamma-subunits of the receptor for immunoglobulin E.

The surface receptor for immunoglobulin E (IgE) on rat basophilic leukemia cells and their normal counterparts has been postulated to consist of four polypeptide chains: a 45-kDa alpha-chain which binds IgE, a 33-kDa beta-component and two disulfide-linked, 9-10-kDa gamma-polypeptides. The instability of this complex in mild detergents makes it possible that, in vivo also, the structure may not be stable and that there is an independent assembly or exchange of the chains. We studied this question using surface-labeling and biosynthetic labeling techniques and found that the chains turn over coordinately and do not independently exchange. The results provide further support for the proposal that the alpha beta gamma 2 complex is the unit receptor for IgE.