The Journal of Allergy and Clinical Immunology
Volume 104, Issue 2 , Pages 492-498, August 1999

Upregulation of FcϵRI on human basophils by IgE antibody is mediated by interaction of IgE with FcϵRI☆☆☆★

Baltimore, Md, and London, United Kingdom

From athe Johns Hopkins Asthma and Allergy Center, Baltimore; and bThe Randall Institute, King’s College London, London

Received 15 March 1999; received in revised form 12 May 1999; accepted 13 May 1999.

Article Outline

Abstract 

Background: IgE is now known to upregulate the expression of FcϵRI on human basophils. It is not known which receptor on basophils mediates this process of upregulation. Objective: We sought to determine whether galectin-3, FcϵRII (CD23), or FcϵRI were involved in the upregulation of FcϵRI by IgE. Methods: The role of galectin-3 was examined by measuring the influence of α-lactose on upregulation. Basophils were examined for expression of FcϵRII (CD23) by flow cytometry and messenger (m)RNA expression. Functional discrimination between binding to FcϵRII or FcϵRI was examined through the use of mutant IgE-Fc fragments or anti-FcϵRII antibody. Results: Upregulation of FcϵRI on basophils in the presence of IgE was not altered by coincubation with α-lactose, eliminating a role for galectin-3. Basophils were not found to express FcϵRII, as determined by flow cytometry with enriched basophil preparations or RT-PCR with highly purified basophil preparations. A mutant of the Fc fragment of IgE (IgE-Fc), which binds to FcϵRI with a greater than 10-fold lower affinity than IgE or wild-type IgE-Fc but exhibits no change in affinity for FcϵRII, allowed us to distinguish between the functions of the two Fc receptors. The mutant (R334S; Henry et al 1997) was required at about 30-fold higher concentration than the wild-type IgE-Fc for the same stimulation of FcϵRI expression on basophils, thus excluding a role for FcϵRII in the response. In addition, treatment of basophils with anti-FcϵRII antibody (MHM6), which is known to be competitive with IgE, had no effect on the expression of FcϵRI or the ability of IgE to upregulate expression of FcϵRI. Conclusion: Collectively, these data indicate that IgE interacts with FcϵRI to upregulate its expression on human basophils. (J Allergy Clin Immunol 1999;104:492-8.)

Keywords:  IgE receptors, basophils, IgE antibody

Abbreviations:  EC50 , Effective concentration for 50% of the maximum effect, PIPES , Piperazine-N,N-bis-2-ethanesulfonic acid, SPR , Surface plasmon resonance

 

Recent studies have demonstrated the regulation of cell surface FcϵRI expression on basophils and mast cells obtained from either mice or humans by IgE antibody.1, 2, 3, 4, 5 In the absence of IgE, cell surface FcϵRIα decreases, and in its presence cell surface FcϵRI accumulates. In either instance the total cellular mass of FcϵRIα also decreases and increases.3 The increase in mass during incubation with IgE indicates the synthesis of FcϵRIα. Inhibition of the increase in FcϵRIα by cycloheximide in studies with mouse mast cells supports this conclusion.4 It is not yet clear whether synthesis is induced or constitutive. Upregulation occurs in purified cell preparations, and therefore it is unlikely that IgE induces upregulation indirectly through another cell type.3 Denaturing IgE with heat (56°C for 90 minutes) or blocking the Cϵ2/Cϵ3 region with specific mAbs blocks its ability to induce upregulation.3 However, the upregulation induced by IgE is slow, and the concentration dependence on IgE does not directly support the conclusion that upregulation occurs through binding of IgE to FcϵRIα. On the basis of the estimates of the equilibrium constant for human IgE interacting through FcϵRIα, one would expect that if upregulation depended on the equilibrium conditions of binding, then the effective concentration for 50% of the maximum effect (EC50) would be between 10 and 50 ng/mL of IgE antibody. In human basophils the EC50 for upregulation occurs at approximately 250 ng/mL, and the concentration dependence is quite broad.3 This result raised some concerns about which IgE-binding receptor might be mediating the effects of IgE. The current studies were performed to test the conclusion that upregulation results from a direct interaction between IgE and FcϵRIα. Three possibilities were explored, namely that IgE interaction occurs through FcϵRI, FcϵRII, or ϵBP (galectin-3). In a previous report we eliminated a fourth possibility for interaction of IgE through CD32 on human basophils by noting that high concentrations of IgG (1 mg/mL) neither induced upregulation of FcϵRI nor altered the upregulation induced by IgE.3

Back to Article Outline

METHODS 

Buffers 

Piperazine-N,N-bis-2-ethanesulfonic acid (PIPES; Sigma Chemical Co, St Louis, Mo) stock buffer (25 mmol/L PIPES containing 110 mmol/L NaCl, 5 mmol/L KCl, and 40 mmol/L NaOH adjusted to pH 7.3) was stored at 10 times the above concentration. We also used PAG (PIPES [1×] containing 0.003% human serum albumin [Miles Laboratories Inc, Elkhart, Ind] and 0.1% glucose), PAGCM (PAG with 1.0 mmol/L CaCl2 and 1.0 mmol/L MgCl2 ), and PAG-EDTA (PAG with 1.0 mmol/L EDTA).

Reagents 

Purified IgE-PS myeloma was a gift from Dr T. Ishizaka.6 Recombinant human IL-3 was obtained from Biosource (Camarillo, Calif). IgE-Fc(WT) and IgE-Fc(R334S) were obtained by procedures previously described and represent materials shown to be free of aggregates by HPLC.7, 8 The so-called IgE-Fc(WT) is only considered a wild type with respect to R334S. Both IgE-Fcs bear 2 mutations, Asn265Gln and Asn371Gln, which were made to make the IgE more homogeneous by eliminating the exposed carbohydrates. The conserved (buried) glycosylation site N394 was retained because it was thought that it may be important in the structure. MHM6 antibody for flow cytometry of FcϵRII was obtained from Dako (Carpintaria, Calif). The messenger (m)RNA primer pair for analysis of FcϵRII mRNA by RT-PCR was obtained from Operon (Alameda, Calif) with the following sequences: 5′-CGTGATGATGCGGGGCTC and 5′-AGAGGAGCGGGAGATGTG. Conditions for using these primers were worked out on Ramos cells (kindly provided by Dr Farhad Imani, Johns Hopkins University Asthma and Allergy Center), which express high levels of this receptor. The PCR procedure used standard reagents (PCR kits; Perkin-Elmer, Foster City, Calif) with thermal cycling set for denaturing (94°C for 45 seconds), annealing (66°C for 45 seconds), and extension (72°C for 45 seconds) for 32 cycles.

Cell preparation 

Two types of basophil preparations were used. Most of the studies used cells obtained from leukapheresis and were prepared as previously described.9 Basophil purity in these preparations ranged from 15% to 95% with a median of 33%. In some experiments cells were isolated from peripheral blood by using the double Percoll method.10 The blood was diluted with EDTA-saline and centrifuged at 500g for 15 minutes to obtain a buffy coat. The buffy coat cells were diluted in saline and layered onto a 2-step Percoll gradient (upper layer, 1.065 g/mL; lower layer, 1.079 g/mL), as described previously.11 After centrifugation at 450g for 15 minutes, the interface between the 1.065 Percoll/plasma upper layer and the 1.079 lower Percoll layer was harvested and washed as above (basophil purity of 8% to 45%). The specific cell preparations used will be noted in the text. One of the preparations used to examine expression of CD23 mRNA in human basophils was performed on basophils further purified by positive selection. The procedure has been described elsewhere.9 Briefly, cells were incubated for 10 minutes with 1 μg/mL mouse anti-human IgE (TES-19; kindly provided by Dr Frances Davis at Tanox Corp) in the presence of 4 mg/mL normal human IgG to block FcγR; the entire procedure was carried at a temperature below 4°C in PAG with 50 μmol/L EDTA. After a subsequent 20-minute incubation with rat anti-mouse IgG2a+b paramagnetic beads (8 μL/107 cells), the cells were passed through a MACS mini-column (MACS system; Miltenyi Biotec Inc, Sunnyvale, Calif). Both flow-through cells (contaminating mononuclear cells) and column-bound cells (basophils) were collected.

Cell culture 

Enriched basophil preparations were cultured in Iscove’s modified Dulbecco’s medium media (Life Technologies) containing 2% FCS, 40 μg/mL gentamycin, and 10 ng/mL IL-3. The total cell density was 2 × 106/mL, and culturing was done in 96-, 24-, or 6-well tissue culture–treated plates (Costar, Cornell, NY) as appropriate.

Flow cytometry 

A flow cytometric technique incorporating light scatter characteristics was used to quantify FcϵRIα chain expression on basophils as described.12 Cell surface expression of FcϵRIα chain was detected by using a mouse IgG1 anti-human FcϵRIα chain mAb (22E7; generously provided by J Kochan, Roche Pharmaceuticals13) and was compared with labeling with an identical concentration of irrelevant mouse IgG1 (Coulter, Hialeah, Fla). The 22E7 antibody has been shown to recognize an epitope that is unaffected by FcϵRIα occupancy.13 Aliquots of cells were labeled in PAG containing 0.2% human serum albumin with 1 mg/mL human IgG to minimize nonspecific binding to FcγR.12 Each of the mAbs were used at concentrations predetermined to be optimal for labeling. Titration of the MHM6 antibody was performed on preparations of lymphocytes or Ramos cells (kindly provided by Dr Farhad Imani, JHU-AAC). Dilutions of 1:100 were used for these experiments. Binding of mAbs was detected by using saturating concentrations of R-phycoerythrin–conjugated polyclonal goat anti-mouse IgG (Tago, Burlingame, Calif). An EPICS Profile flow cytometer (Coulter, Inc) was used to analyze fluorescent signals after excitation at 488 nm. “Bitmap” gates, which are intermediate between the forward and side scatter characteristics of lymphocytes and monocytes, were used to select for a population of cells that were predominantly basophils. Because the cells were already enriched in basophils, these bitmaps can select a population of cells that is generally greater than 80% basophils, with the primary contaminants being lymphocytes. Data are expressed as the mean fluorescence in labeled cells minus the mean fluorescence of IgG1 controls. Day-to-day variability in the sensitivity of the flow cytometer was corrected by noting or adjusting the photomultiplier tube voltage to generate the same signal for a set of standard calibration beads (Immunochek, Coulter). For the analysis of MHM6 binding, the basophil preparations were also incubated with rabbit FITC-labeled anti-IgE antibody, and the bitmap selected cells also gated on green fluorescence to focus on cells that were exclusively basophils. The photomultiplier tube voltages for red fluorescence (phycoerythrin staining) were set very high so that a full distribution of IgG1 control antibody staining could be appreciated. This high setting should also enhance the likelihood of detecting low levels of binding (eg, with the settings used for basophils, Ramos cells were found to give off-scale distributions), although these settings also introduced considerable noise.

In previously published studies the flow cytometric measurements were calibrated, examining the fluorescence staining of 6 donors’ basophils that spanned a moderate range of staining intensities (7 to 120 fluorescent units or 8000 to 140,000 FcϵRI per basophil) and simultaneously assessing receptor or IgE density by the acetate elution method. 22E7 staining (ordinate) compared with total FcϵRI density by acetate elution (after sensitizing with PS myeloma IgE as described above) was linear, with a slope of 0.00084 (ie, a fluorescence measurement of 100 represents approximately 120,000 receptors) and an r value of 0.963.

Statistical analysis 

The Student t test was used for most statistical comparisons, whereas other comparisons were done with a nonparametric Wilcoxon signed-rank statistic or ANOVA. If errors or error bars are shown, they represent SEM unless otherwise indicated.

Back to Article Outline

RESULTS 

A role for galectin-3 (ϵBP) was excluded by performing upregulation experiments in the presence or absence of α-lactose. Fig 1 demonstrates the absence of an effect of 25 mmol/L lactose on upregulation, a concentration which should completely inhibit the interaction of IgE oligosaccharide chains with galectin-3.14, 15

  • View full-size image.
  • Fig. 1. 

    Effect of α-lactose on the IgE-mediated upregulation of FcϵRI on human basophils. Enriched basophils (average of 46% purity) were cultured for 7 days in the presence or absence of IgE antibody at 1 μg/mL and in the presence or absence of α-lactose at 25 mmol/L (n = 3). Cell surface FcϵRI was detected by flow cytometry by using mAb 22E7. The control mean fluorescent intensity was 28 ± 5 units.

Interaction with FcϵRII was examined by 4 different approaches. First, basophils were cultured with 2 fragments of the IgE molecule generated by recombinant techniques. Both fragments contain the epsilon second through fourth constant regions (Cϵ2 to Cϵ4) as a disulphide-linked dimer, which is referred to as the IgE-Fc, but they differ in that the IgE-Fc(R334S) has the arginine at residue 334 in the wild-type sequence replaced by serine.8 The FcϵRI binding characteristics, as determined in cell-binding and surface plasmon resonance (SPR) assays, have been previously published,8 and the results are summarized in Table I. The important distinguishing characteristic is that IgE-Fc(R334S) has a significantly lower affinity for FcϵRI than IgE-Fc(WT) while retaining its native affinity for FcϵRII. The relative decrease in binding affinity of IgE-Fc(R334S) compared with IgE-Fc(WT) differed between the cell-binding assay (a factor of 15) and the SPR assay, which displayed biphasic binding kinetics (a factor of 12 for Ka1 and a factor of 120 for Ka2 ) but can be regarded as a difference greater than 10-fold in all assays. The previously published measurements of Ka for FcϵRII in a cell binding assay (Table I) have been confirmed by SPR and show that the mutation had no effect on binding to this receptor. Fig 2 shows the concentration dependence of FcϵRI upregulation on basophils cultured with either IgE-Fc(WT) or IgE-Fc(R334S).

  • View full-size image.
  • Fig. 2. 

    Upregulation of FcϵRI on basophils cultured in the presence or absence of IgE-Fc(WT) or IgE-Fc(R334S) (n = 4). Basophils were cultured with several concentrations of either fragment, noted on the abscissa, for 7 days. Upregulation is expressed as the ratio of FcϵRI expression on day 7 to its expression on day 1 as detected by flow cytometry with 22E7. The starting average mean fluorescent intensity for 22E7 staining was 25 ± 3 units, which is similar to the starting FcϵRI densities for the previously published experiments with PS myeloma IgE (see “Methods” section).3

These data show that there is an approximately 30-fold difference in EC50 for upregulation of FcϵRI, as would be expected if IgE were interacting with FcϵRIα.

Table I. Previously published binding constants for recombinant IgE-Fc binding to FcϵRI or FcϵRII
Type of assayReceptorConstantIgE-Fc(WT)IgE-Fc(R334S)
Cell bindingFcϵRIkf9.9 ± 1.1 × 1057.5 ± 2.6 × 105
kr1.7 ± 0.2 × 10–52.0 ± 0.1 × 10–4
Ka5.8 × 10103.7 × 109
SPRFcϵRIkf11.2 ± 0.8 × 1063.9 ± 3.2 × 105
kf22.5 ± 1.8 × 1059.3 ± 3.8 × 104
kr11.0 ± 0.1 × 10–24.6 ± 0.4 × 10–3
kr22.0 ± 0.1 × 10–49.9 ± 0.4 × 10–3
Ka11.1 × 1088.6 × 106
Ka21.2 × 1091.0 × 107
Cell bindingFcϵRIIKa3.2 ± 2.4 × 1072.4 ± 3.9 × 107

Whereas the R334S mutation in IgE-Fc(R334S) might not have altered the binding affinity for FcϵRII, this mutation might alter any functional consequence of this binding. Therefore additional studies were carried out to test for FcϵRII expression on human basophils. Basophils are not known to express CD23, as tested by flow cytometry. However, flow cytometry is not a sensitive technique when used on basophils obtained by dextran sedimentation or single step density gradients (purities <2%); expression levels less than 5000 molecules per cell would not be detected.12 However, by using more enriched basophil preparations (>20%), flow cytometry is somewhat more sensitive and able to detect IgE densities at around 1000 per cell. Therefore partially enriched basophil preparations were tested for FcϵRII expression by using MHM6 antibody. As demonstrated in Fig 3, there was no difference in the flow cytometric distributions when control mouse IgG or MHM6 were used as the primary antibodies.

  • View full-size image.
  • Fig. 3. 

    Lack of FcϵRII expression on the basophil cell surface as detected by flow cytometry with MHM6. Enriched basophil preparations (average purity of 35% ± 10%) were examined by flow cytometry by using a 1:100 dilution of MHM6. The left panel shows the results for one experiment. The right panel shows the average of results from 9 different preparations of basophils, with the data expressed as the ratio of mean fluorescence for cells incubated with MHM6 versus control antibody (irrelevant mouse IgG at 50 μg/mL).

Fig 4 demonstrates that FcϵRII could not be detected in basophil mRNA preparations examined by RT-PCR.

  • View full-size image.
  • Fig. 4. 

    RT-PCR of mRNA derived from purified basophils or contaminating cells from the same preparations (see “Methods” section). The left side of the figure shows results for basophils, with 3 dilutions of the original sample, and the right side of the figure shows the results for the contaminating cells. The purity of the basophil preparations is noted on the left (n = 3).

Basophils were purified to near homogeneity by positive selection, and their mRNA was extracted. The mRNA from contaminating cells remaining after positive selection was also examined after extraction. Fig 4 shows 3 preparations of purified basophils compared with the contaminating cells. The right halves of the gels show the results for serially diluted samples of the contaminating cell mRNA. The left halves of the gels show results for serially diluted samples from basophils. In 2 of the lower purity basophil preparations (95% and 94%), a close inspection reveals a faint band in the lane representing undiluted samples, the density of which is consistent with the expected density of a band derived from the contaminating cells diluted to this extent. In data not shown, the house-keeping mRNA for hypoxanthine phosphoribosyl transferase was readily detected in both samples obtained from basophils and contaminating cells by using the appropriate primers.

MHM6 antibody has been found to induce biologic responses in systems in which FcϵRII is known to play a role,16, 17, 18, 19, 20 and it is known to be competitive with IgE.21 Therefore we examined whether this antibody could influence the upregulation of FcϵRI on basophils in a 7-day culture with or without IgE antibody. As shown in Fig 5, MHM6 caused no significant changes in the expression of FcϵRIα at any of the concentrations tested; no effect on FcϵRIα expression in the absence or presence of IgE was observed.

  • View full-size image.
  • Fig. 5. 

    Effect of treating basophils with MHM6 antibody. Basophils were cultured with or without IgE antibody at 1 μg/mL in the presence or absence of several dilutions of MHM6 antibody. After 7 days of culture, the cells were harvested and analyzed by flow cytometry with 22E7. Data are expressed in arbitrary fluorescence units (n = 3).

Parenthetically, although the absolute concentration of the commercial MHM6 antibody is not known, as noted in the flow cytometric studies (see “Methods” section), titration of the antibody showed that a 1:100 to 1:300 concentration provided optimal labeling of Ramos cells.

Back to Article Outline

DISCUSSION 

Collectively, these data indicate that there is no interaction of IgE with either galectin-3 or FcϵRII. There are indications that galectin-3 is expressed in human basophils,22 although current thinking suggests that if some of this galectin-3 is found outside the cell, it is probably bound to cell surface carbohydrates by its lectin-binding domain. If so, it is more difficult to envision how it could also then interact with extracellular IgE. Nevertheless, we sought and found a more definitive test of galectin-3 involvement in the upregulation of FcϵRI. The lack of inhibition by α-lactose excludes any such involvement because α-lactose at the concentrations used prevents binding to IgE.14, 15, 23

Four methods were used to test the likelihood that IgE interacts with FcϵRII on basophils. No evidence could be found for basophils expressing even low levels of FcϵRII. Although the RT-PCR procedure detected a faint band of mRNA from purified basophil preparations, the intensity of the band from undiluted samples was consistent with it coming from the small number of contaminating cells in these basophil preparations. Indeed, the band was somewhat fainter than if 5% to 10% of contaminating cells were contributing to its presence, based on the dilutional RT-PCR data shown in the right half of Fig 4. Flow cytometric data and functional data obtained by using MHM6 supported this conclusion. Finally, the difference in the ability of IgE-Fc(WT) and IgE-Fc(R334S) to upregulate FcϵRI expression was consistent with IgE interacting with FcϵRIα because the 30-fold difference agrees with the observed difference in affinity of IgE-Fc R334S and wild-type for FcϵRIα.

The conclusion that IgE antibody interacts with FcϵRIα leads back to the original problem: the concentration-dependence for upregulation appears shifted to concentrations of IgE antibody that are higher than expected from a simple assumption of equilibrium binding between IgE and FcϵRIα. Expectations for these results were based on binding constants for IgE to basophil FcϵRI, which we have determined in recent in vitro studies.3 However, these values are not reflective of binding constant values found in the literature. The dissociation constant determined in our recent studies of IgE dissociation from basophils in culture3 was found to be low (of the order of 1 × 10–6 sec–1) compared with other published studies.24, 25, 26, 27 In addition, our calculations of the forward binding constants have been consistently lower than other published values.3, 24, 25, 26, 27, 28 The source of the discrepancies is not currently known. Therefore a strong conclusion regarding an expected EC50 for upregulation of FcϵRI by IgE cannot be made; that is, the value of 250 ng/mL may very well be accounted for by a particular choice of published rate constants for human IgE antibody and human FcϵRI. However, it is useful to note that we are currently exploring a model of this upregulation that predicts that the relatively slow binding of IgE to the receptor (regardless of which forward binding constants one accepts as correct) could cause an apparent requirement for higher concentrations of IgE to induce upregulation. The central tenets of the model are that the receptor is constitutively synthesized and that unoccupied receptor is susceptible to loss from the cell surface. This is akin to the currently accepted model of FcϵRII expression in which a metalloprotease has been identified and shown to mediate the loss of unoccupied FcϵRII from the cell surface.29 Relatively specific inhibitors of this enzyme can be shown to upregulate expression of FcϵRII. Future experiments will be needed to verify the basic tenets of a similar model for FcϵRI regulation, namely that synthesis is constitutive rather than induced and that only unoccupied receptor is lost from the cell surface. The mechanism of this loss also needs exploration, and future experiments need to determine whether loss is a saturable process. In summary, our results indicate that IgE interacts with FcϵRI to upregulate its expression, but the precise explanation for the in vitro results concerning concentration dependence awaits further study.

Back to Article Outline

References 

  1. Hsu C, MacGlashan DW. IgE antibody up-regulates high affinity IgE binding on murine bone marrow derived mast cells. Immunol Lett. 1996;52:129–134
  2. MacGlashan DW, Bochner BS, Adelman DC, Jardieu PM, Togias A, Mckenzie-White J, et al.  Down-regulation of FcϵRI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody. J Immunol. 1997;158:1438–1445
  3. MacGlashan DW, White-Mckenzie J, Chichester K, Bochner BS, Davis FM, Schroeder JT, et al.  In vitro regulation of FcϵRIα expression on human basophils by IgE antibody. Blood. 1998;91:1633–1643
  4. Yamaguchi M, Lantz CS, Oettgen HC, Katona IM, Fleming T, Miyajama I, et al.  IgE enhances mouse mast cell FcϵRI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions. J Exp Med. 1997;185:663–672
  5. Lantz CS, Yamaguchi M, Oettgen HC, Katona IM, Miyajama I, Kinet JP, et al.  IgE regulates mouse basophil FcϵRI expression in vivo. J Immunol. 1997;158:2517–2521
  6. Ishizaka K, Ishizaka T. Biological function of gamma E antibodies and mechanisms of reaginic hypersensitivity. Clin Exp Immunol. 1970;6:25–42
  7. Young RJ, Owens RJ, Mackay GA, Chan CM, Shi J, Hide M, et al.  Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants. Protein Eng. 1995;8:193–199
  8. Henry AJ, Cook JP, McDonnell JM, Mackay GA, Shi J, Sutton BJ, et al.  Participation of the N-terminal region of Cϵ3 in the binding of human IgE to its high-affinity receptor FcϵRI. Biochemistry. 1997;36:15568–15578
  9. MacGlashan DW, White JM, Huang SK, Ono SJ, Schroeder J, Lichtenstein LM. Secretion of interleukin-4 from human basophils: the relationship between IL-4 mRNA and protein in resting and stimulated basophils. J Immunol. 1994;152:3006–3016
  10. Schroeder JT, MacGlashan DW, Kagey-Sobotka A, White JM, Lichtenstein LM. The IgE-dependent IL-4 secretion by human basophils: the relationship between cytokine production and histamine release in mixed leukocyte cultures. J Immunol. 1994;153:1808–1817
  11. Warner JA, Reshef A, MacGlashan DW. A rapid Percoll technique for the purification of human basophils. J Immunol Methods. 1987;105:107–110
  12. Bochner BS, McKelvey AA, Schleimer RP, Hildreth JE, MacGlashan DW. Flow cytometric methods for the analysis of human basophil surface antigens and viability. J Immunol Methods. 1989;125:265–271
  13. Riske F, Hakimi J, Mallamaci M, Griffin M, Pilson B, Tobkes N, et al.  High affinity human IgE receptor (FcϵRI): analysis of functional domains of the α-subunit with monoclonal antibodies. J Biol Chem. 1991;266:11245–11251
  14. Wollenberg A, de la Salle H, Hanau D, Liu FT, Bieber T. Human keratinocytes release the endogenous beta-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms. J Exp Med. 1993;78:777–785
  15. Hsu DK, Zuberi RI, Liu FT. Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin. J Biol Chem. 1992;267:14167–14174
  16. Flores-Romo L, Johnson GD, Ghaderi AA, Stanworth DR, Veronesi A, Gordon J. Functional implication for the topographical relationship between MHC class II and the low-affinity IgE receptor: occupancy of CD23 prevents B lymphocytes from stimulating allogeneic mixed lymphocyte responses. Eur J Immunol. 1990;20:2465–2469
  17. Bettler B, Maier R, Ruegg D, Hofstetter H. Binding site for IgE of the human lymphocyte low-affinity Fcϵ receptor (FcϵRII/CD23) is confined to the domain homologous with animal lectins. Proc Natl Acad Sci USA. 1989;86:7118–7122
  18. Goff LK, Armitage RJ, Beverley PC. Characterization of two CD23 monoclonal antibodies with reactivity distinct from other antibodies within this cluster of differentiation. Immunology. 1988;65:213–220
  19. Paul-Eugene N, Kolb JP, Abadie A, Gordon J, Delespesse G, Sarfati M, et al.  Ligation of CD23 triggers cAMP generation and release of inflammatory mediators in human monocytes. Immunology. 1992;149:3066–3071
  20. Dugas N, Vouldoukis I, Becherel P, Arock M, Debre P, Tardieu M, et al.  Triggering of CD23b antigen by anti-CD23 monoclonal antibodies induces interleukin-10 production by human macrophages. Eur J Immunol. 1996;26:1394–1398
  21. Gordon J, Webb AJ, Guy GR, Walker L. Triggering of B lymphocytes through CD23: epitope mapping and studies using antibody derivatives indicate an allosteric mechanism of signalling. Immunology. 1987;60:517–521
  22. Craig SS, Krishnaswamy P, Irani AM, Kepley CL, Liu FT, Schwartz LB. Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils. Anat Rec. 1995;242:211–219
  23. Laing JG, Robertson MW, Gritzmacher CA, Wang JL, Liu FT. Biochemical and immunological comparisons of carbohydrate-binding protein 35 and an IgE-binding protein. J Biol Chem. 1989;264:1097–1110
  24. Ishizaka T, Helm B, Hakimi J, Niebyl J, Ishizaka K, Gould H. Biological properties of a recombinant human immunoglobulin epsilon-chain fragment. Proc Natl Acad Sci USA. 1986;83:8323–8327
  25. Ishizaka T, Dvorak AM, Conrad DH, Niebyl JR, Marquette JP, Ishizaka K. Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells. J Immunol. 1985;134:532–540
  26. Miller L, Blank U, Metzger H, Kinet JP. Expression of high-affinity binding of human immunoglobulin E by transfected cells. Science. 1989;244:334–337
  27. Hakimi J, Seals C, Kondas JA, Pettine L, Danho W, Kochan J. The alpha subunit of the human IgE receptor (FcϵRI) is sufficient for high affinity IgE binding. J Biol Chem. 1990;265:22079–22081
  28. Goldstein B, Dembo M, Malveaux FJ. Some quantitative aspects of the passive sensitization of human basophils. J Immunol. 1979;122:830–833
  29. Marolewski AE, Buckle DR, Christie G, Earnshaw DL, Flamberg PL, Marshall LA, et al.  CD23 (FcϵRII) release from cell membranes is mediated by a membrane-bound metalloprotease. Biochem J. 1998;333(Suppl):573–579

☆ Supported by National Institutes of Health grants AI20253 and AI42220.

☆☆ Reprint requests: Donald MacGlashan, Jr, MD, PhD, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.

★ 0091-6749/99 $8.00 + 0  1/1/99999

PII: S0091-6749(99)70399-4

The Journal of Allergy and Clinical Immunology
Volume 104, Issue 2 , Pages 492-498, August 1999

Article Tools

* Image Usage & Resolution