Ige Receptors on Human Basophils. Relationship to Serum Ige Concentration
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IgE receptors on human basophils. Relationship to serum IgE concentration. F J Malveaux, … , N F Adkinson Jr, L M Lichtenstein J Clin Invest. 1978;62(1):176-181. https://doi.org/10.1172/JCI109103. Research Article As reported previously, and confirmed here in 26 donors, the serum IgE level (2.6-5,500 ng/ml) correlates well (rs = 0.95, P less than 0.001) with the in vivo number of IgE molecules/basophil (6,000-600,000). The total number of IgE receptors/basophil was monitored by incubating them with an IgE-rich serum (15 microgram/ml), quantitatively stripping IgE from the cells at pH 3.7, and measuring eluted IgE by a direct radioimmunosorbent test. Saturation of receptors for each donor was achieved with 15 nM IgE (3 microgram/ml). The proportion of receptors occupied in vivo correlated with the serum IgE (rs = 0.84, P less than 0.001) whereas the average association constant of the receptors was independent of serum IgE and ranged from 7.1 X 10(8)/M to 2.8 X 10(10)/M, averaging 7.7 X 10(9)/M. Unexpectedly, the total number of IgE receptors/basophil was closely related to the serum IgE level. (rs = 0.92, P less than 0.001). Thus, either there is genetic association between serum IgE and the number of basophil IgE receptors, or, more likely, the receptor number is modulated by the serum IgE concentration. Find the latest version: https://jci.me/109103/pdf IgE Receptors on Human Basophils RELATIONSHIP TO SERUM IgE CONCENTRATION FLOYD J. MALVEAUX, MARY CAROL CONROY, N. FRANKLIN ADKINSON, JR., and LAWRENCE M. LICHTENSTEIN, Department of Medicine, Division of Clinical Immunology, the Johns Hopkins University, School of Medicine, Baltimore, Maryland 21239 A B S T RA C T As reported previously, and confirmed the release of histamine from human basophils is inde- here in 26 donors, the serum IgE level (2.6-5,500 pendent of the number of IgE molecules on their sur- ng/ml) correlates well (r, = 0.95, P < 0.001) with the in face (1). vivo number of IgE molecules/basophil (6,000- This manuscript describes additional studies aimed 600,000). The total number of IgE receptors/basophil at direct assessment of the following additional param- was monitored by incubating them with an IgE-rich eters: (a) the average association constant (Ko)l of the serum (15 ,ug/ml), quantitatively stripping IgE from the interaction between the Fc of IgE and the basophil cells at pH 3.7, and measuring eluted IgE by a direct IgE receptor; (b) the degree of receptor saturation radioimmunosorbent test. Saturation of receptors for in vivo; and (c) the total number of IgE receptors/ each donor was achieved with 15 nM IgE (3 ,ug/ml). basophil. 26 unselected donors were evaluated with re- The proportion of receptors occupied in vivo corre- gard to serum IgE concentration and the number of IgE lated with the serum IgE (r, = 0.84, P < 0.001) whereas molecules/basophil in vivo. Their basophils were then the average association constant of the receptors was passively saturated with a high IgE serum, and the independent of serum IgE and ranged from 7.1 x 108/ increment in cell-bound IgE measured by elution at M to 2.8 x 10'0/M, averaging 7.7 x 109/M. Unex- low pH. Among the individuals studied, there was a 10- pectedly, the total number of IgE receptors/basophil to 100-fold range in the calculated Ko values for the was closely related to the serum IgE level. (r8 = 0.92, basophil IgE receptors. The study further showed a P < 0.001). Thus, either there is genetic association correlation between basal serum IgE and IgE recep- between serum IgE and the number of basophil IgE tors/basophil. This finding suggests either that the two receptors, or, more likely, the receptor number is traits are genetically associated or that there is physio- modulated by the serum IgE concentration. logical modulation of basophil receptor display by cir- culating IgE. INTRODUCTION This laboratory has recently been concerned with METHODS understanding, in quantitative terms, the IgE com- Materials. Tris-EDTA buffer contained 0.025 M Tris, ponents of the mediator-release process from human 0.12 M NaCl, 0.005 M KCI, 0.03% human serum albumin, basophils (1). By measuring the IgE eluted from and 0.01 M EDTA. The acetate buffer used for elution con- sisted of 0.05 M acetate, 0.085% NaCl, 0.01 M EDTA, and washed leukocytes at low pH, a technique first de- 0.03% human serum albumin. This buffer was adjusted to scribed by Ishizaka and Ishizaka (2), we previously pH 3.7 at 0°C. Goat antiserum against the Fc fragments of reported a strong positive correlation between total IgE myeloma protein (PS) was generously supplied by Dr. serum and cell-bound IgE on human basophils (1). We Kimishige Ishizaka (Johns Hopkins University at Good have also observed, using anti-IgE as a stimulus, that Samaritan Hospital). IgE-rich serum was obtained from a gg/ml. The AB serum used for diluting the reaginic serum was obtained from a normal donor whose serum IgE was Dr. Malveaux is the recipient of National Research Service 25 ng/ml. Award Al 05385 from the National Institutes of Health. Dr. Preparation of cells. Venous blood collected from each Conroy's present address is Ciba-Geigy Pharmaceutical Cor- donor was mixed with 6% dextran 70 in saline (Cutter Labora- poration, Ardsley, N. Y. 10502. Dr. Adkinson is the recipient tories, Inc., Berkeley, Calif.), 0.01 M EDTA, and 350 mg of Allergic Disease Academic Award Al 71023 from the National Institutes of Health. Received for publication 22 August 1977 and in revised I Abbreviations used in this paper: Ko, average association form 9 March 1978. constant; r, proportion of receptors occupied. 176 J. Clin. Invest. © The American Society for Clinical Investigation, Inc., 0021-9738/78/0701-176 $1.00 dextrose and allowed to sediment for 60-90 min at room nM IgE temperature (3). The leukocyte-rich plasma was centrifuged 0.016 0.66 2 6 15 30 and the cell button was washed twice in an equal volume 20- of Tris-EDTA buffer. The cells were suspended in 6.0 ml * Preelultion Fraction CA) Tris-EDTA and the were counted in tripli- Cl Elution Froction ("B') buffer basophils Ist Postelution Wash z * ('C") cate in a Spiers-Levy Chamber (Hausser Scientific Co., Blue 0 (JJ 2nd Postelution Wash( D") Bell, Pa.) after staining with Alcian blue according to P- 15- the method of Gilbert and Ornstein (4). To 50 ,ul of the tr cell suspension mixed with 0.45 ml of 0.1% EDTA in L(. w saline was added 0.45 ml of a solution containing 0.076% a. a 10- acetyl pyridinium chloride, 0.7% lanthanum chloride, w 0.9% sodium chloride, 0.21% Tween 20 (Atlas Chemical -jcr Industries, Inc. Wilmington, Del.), and 0.143% Alcian blue. Li After agitation of this mixture for 60 s, 50 ,ul of 1 N HCI was added and an aliquot was transferred to the Spiers-Levy 5- counting chamber. The actual number of basophils counted for each donor ranged from 200 to >1,200. The mean coeffi- cient of variation for this method of basophil counting L/Z I t MEu was 14%. 0.003H 0.13 0.40 1.20 3.00 6.00 Passive sensitization. The method of sensitization was Ig E (ug/ml) DURING SENSITIZATION that described by Levy and Osler (5). Briefly, 107_108 washed leukocytes (from 30 to 50 ml blood) suspended in FIGURE 1 Quantity of IgE measured in the supernatant 1.0 ml of Tris-EDTA buffer were combined with 0.5 ml fluids at different steps of the experimental protocol after heparin-EDTA and 1.0 ml of an appropriate dilution (in AB passive sensitization. Sensitization at 3 ng/ml was carried serum) of a single IgE-rich reaginic serum. An equal number out in autologous serum. (A) Preelution with Tris-EDTA of washed leukocytes was suspended in a comparable mix- (pH 7.4) at 0°C for 10 min; (B) Acetate-EDTA (pH 3.7) elution ture containing autologous serum. Incubation proceeded at 0°C for 10 min; (C and D) Postelution washings with for 90 min at 37°C on a reciprocal shaker. Tris-EDTA. Elution of IgE. After sensitization, leukocytes were washed twice with 5 ml Tris-EDTA buffer, suspended in 1 ml of the same buffer (chilled to 0°C), and allowed to sit 10 min formed to establish whether human basophils could at 0°C (fraction A). After centrifugation, the cells were be saturated with IgE. Fig. 2 shows the results when resuspended in 1 ml of elution buffer, pH 3.7, for an addi- the cells of three individuals were incubated with in- tional 10 min at 0°C (fraction B). These cells were centri- creasing concentrations of the same IgE-rich serum. fuged and the supernatant fluid was immediately titrated to neutrality with 1 N NaOH. The acid elution technique The elutable IgE increases with increasing IgE con- was reported in detail by Ishizaka and Ishizaka (2). Two centrations until a plateau is reached. Apparent satura- subsequent washings were carried out in 1.0 ml Tris-EDTA tion of receptors occurs at about 15 nM [3 jig/ml] IgE buffer (fractions C and D). All supernatant fluids were for each donor. This was later shown to be independent frozen for subsequent total IgE determination.