characterization of prostaglandin (pg)-binding sites expressed on

THE JOURNAL OF BIOLOGICAL CHEMISTRY (a 1992hy The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 18, Issue of June 25, pp. 12700-12708,1992 Printed in U.S.A.

Characterization of Prostaglandin (PG)-binding Sites Expressed on Human Basophils EVIDENCE FOR A PROSTAGLANDIN El, Iz, AND A Dz RECEPTOR*

(Received for publication, September 5, 1991)

Irene Virgolini$j, Shuren Lis, Christian SillaberV, Otto MajdicII , Helmut Sinzinger$, Klaus LechnerlI, Peter Bettelheim7, and Peter Valent7 From the $Department of Nuclear Medicine and Ludwig Boltzmann Institute for Nuclear Medicine, 7Z. Medical Department and the Illnstitute of Immunology, University of Vienna, A-1090 Vienna, Austria

Recent data suggest that prostaglandins (PGs) are involved in the regulation of basophil activation. The aim of this study was to characterize the basophil PG- binding sites by means of radioreceptor assays using 3H-labeled PGs. Scatchard analysis for pure (>95%) chronic myeloid leukemia (CML) basophils revealed two classes of PGEl-binding sites differing in their affinity for the natural ligand (Bmaal = 217 f 65 fmol/

10' cells; Kdz = 47 f 20 nM; ICS0 = PGEl < PG12 < PGD2 < PGEz < PGFz,) as well as two classes of PGIz (i1oprost)-binding sites (Bmaxl = 324 f 145 fmol/lO'

2 7 f 6 nM; ICS0 = PGIz < PGEl < PGD2 < PGEz PGF2,. In addition, CML basophils exhibited a single class of PGDz-binding sites (B,,, = 378 f 98 fmol/lO'

PGEz < PGF2,). In contrast, we were unable to detect specific saturable PGEz-binding sites. Primary and im- mortalized (KU812) CML basophils revealed an identical pattern of PG receptor expression. Basophils (KU812) expressed significantly ( p < 0.001) lower number of PGEl (PGIz)-binding sites (Bmaxl: 9% (20%) of control; Bmexz: 36% (50%) of control) when cultured with recombinant interleukin 3 (rhIL-3), a basophil- activating cytokine, whereas rhIL-2 had no effect on PG receptor expression. Functional significance of binding of PGs to basophils was provided by the dem- onstration of a dose-dependent increase in cellular cAMP upon agonist activation, with PGEl (ED60 = 1.7 f 1.1 nM) and PGIz (ED60 = 2.8 f 2.3 nM) being the most potent compounds. These findings suggest that human basophils express specific receptors for PGEl, PG12 as well as for PGDz.

10' cells; KdI = 0.5 2 0.2 nM; Bmaa2 = 2462 f 381 fmol/

Cells; = 0.5 f 0.3 nM; Bmax2 = 2541 f 381; Kdz =

cells; Kd = 13 f 4 nM; ICSO: PGDz < PGIz < PGE1 <

Basophils are circulating effector cells of allergic reactions (1-4). They produce and/or secrete mediator molecules upon induction with a variety of agonists (5-7). The ability to respond to an agonist depends on expression of specific cell surface receptors and the functional shape of the cell. Human

* This work was supported by a grant from the Onkologie-For- schungsfond of the Medical Facility of the University of Vienna and Grant P7891 from the Fonds zur Forderung der Wissenschaftlichen Forschung in Osterreich. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed Dept. of Nuclear Medicine, University of Vienna, Garnisongasse 13, A-1090 Vienna, Austria. Tel.: 0222-40400-2141; Fax: 43-1-43865214.

blood basophils express a number of agonist-specific receptors (8). The most significant is the high affinity receptor for IgE (9, 10). This molecule is involved in allergen binding and subsequent degranulation (11-13). Other receptors expressed on human basophils may also be involved in basophil activation (14-20). Of particular functional significance are receptors regulating the capacity of the basophils to respond to degranulating compounds. Such receptors are activated either by regulators which enhance the releasability of the basophils, such as interleukins (IL)' (14-16), or by agonists which reduce the releasability of the basophils such as PGs (17-21), corti- costeroids (22), or histamine (23).

A number of observations have suggested that the cAMP system is involved in the regulation of basophil releasability (17-19, 24). In particular, factors potentiating the releasability of human basophils usually lead to a decrease in cAMP levels, and factors which reduce the capacity to degranulate usually promote expression of cAMP (4). However, the exact mechanisms involved in agonist-dependent modulation of basophil releasability still remain unknown.

PGs regulate the functional properties of many cell types including a variety of circulating immune cells (17-21, 23- 28). Many of these effects apparently are mediated via specific cell surface receptors (29-31), while others may involve accessory cells. Previous studies have shown that PGs of the E series reduce the capacity of human blood basophils to respond to degranulating stimuli, whereas PGs of the F series as well as PGDz had either negligible effects or caused poten- tiation of the reaction (5, 17-21, 24). PGs of the E series were also found to increase the levels of cellular cAMP in the effector cells (5). Some of the above mentioned studies utilized rather pure populations of human blood basophils. It has therefore been hypothesized that human blood basophils express PG-binding sites. The cellular substrate, however, remained to be characterized. We have now used a radioreceptor assay to quantitatively analyze the PG-binding sites expressed on highly enriched populations of human basophils.

EXPERIMENTAL PROCEDURES

Materials-['HIPGs were obtained from Amersham International (Buckinghamshire, United Kingdom). The specific activities of the labeled PGs were as follows: ['H]iloprost, 14.1 Ci/mmol; ['HH]PGE,; 42 Ci/mmol; ('H]PGE2, 143 Ci/mmol; [3H]PGD2, 193 Ci/mmol. Ra- diochemical purity was >96% in all preparations as assessed by high performance liquid chromatography on a 5-pm Techsphere ODS

The abbreviations used are: IL, interleukin; PG(s), prostaglandin(s); rhIL-2, recombinant human interleukin 2; rhIL-3, recombinant human interleukin 3; mAb, monoclonal antibodies; CML, chronic myeloid leukemia; PIPES, 1,4-piperazinediethanesulfonic acid; IP, prostaglandin I; EP, prostaglandin E.

12700

Prostaglandin Receptors on Human Basophils 12701

column using a methano1:water:acetic acid gradient. Unlabeled PGL, PGE,, PGE,, 16,16-dimethyl-PGE,, PGD,, and PGF,, were purchased from the Upjohn Co. (Kalamazoo, MI). Unlabeled iloprost (ZK 36374), cicaprost (ZK 96480), and 9-deoxy-9~-chloro-16,17,18,19,20- pentanor-15-cyclohexyl-PGF2,, (ZK 110 841) were kindly provided by Drs. E. Schillinger, C. S. Sturzebecher, and K. K. Thierauch, Schering AG, Berlin, Federal Republic of Germany. Unlabeled 13,14-dihydro- PGE, was a generous gift from Dr. Waltraud Rogatti, Schwarz Pharma, Monheim, Federal Republic of Germany, and unlabeled 6a- carba-PG12 from Dr. S. Moncada, Wellcome Research Laboratories, United Kingdom. All PGs (unlabeled and labeled) were stored at -20 "C. Lyophilized unlabeled PGs were always freshly dissolved in 50 mM Tris-HC1 buffer, pH 9.0, at a stock concentration of 1 mM. No difference in bioactivity between labeled and unlabeled PGs was found as assessed by platelet aggregation studies using ADP and collagen as aggregating stimuli.

Recombinant human (rh) interleukin-3 (IL-3), expressed in Esch- erichia coli, was provided by the Genetics Institute (Cambridge, MA). Purified rhIL-3 had a specific activity of 4.6 X lo6 units/mg of protein as determined by a myeloblast bioassay described by Griffin et al. (32). rhIL-2 was provided by Sandoz Pharma (Vienna, Austria).

A number of mAbs was used to purify CML basophils. The following mAbs were produced at the Institute of Immunology, University of Vienna, and kindly provided by Dr. W. Knapp: VIM13 (CD14) (33), VIBC5 (CD24) (34), VIT3 (CD3) (35), VIMD5 (CD15) (36), VIEG4 (antiglycophorin A) (37), VIT6 (CD1) (38), VIDl (anti-HLA- DR) (39). The following mAbs were purchased Leul (CD5), Leu7 (CD57), and Leu9 (CD7) from Becton Dickinson (Sunnyvale, CA); BMA 022 (anti-HLA-DR) and BMA 0110 (CD2) from Behring (Mar- burg, Federal Republic of Germany), and mAb E-124-2-8 (anti-IgE) from Immunotech (Marseille, France). The mAb CLB-Ery3 (anti- blood group H) was a generous gift from Dr. P. A. T. Tetteroo (Amsterdam).

Purification of CML Basophils-Blood basophils from two CML patients were enriched to near homogeneity as described previously (40, 41) after informed consent was given. Briefly, mononuclear cells were isolated from peripheral venous blood by gradient density centrifugation with Ficoll (density 1.077 g/ml). 5 X lo9 mononuclear cells were incubated in RPMI 1640 medium containing 1 mg of mAb VIMD5 at 4 "C for 45 min. After washing, cells were exposed to 50 ml of rabbit complement (Behring AG, Marburg, Federal Republic of Germany) at 37 "C for 90 min. Washed cells were then exposed to a mixture of mAbs (VIT3, VIBC5, VIM13, Leul, Leu7, Leu9, VIEG4, BMAO1110, BMA022, CLB-Ery3, and VIM-D5; 25 pg/108 cells for each mAb) a t 4 "C for 45 min and then to rabbit complement for another 90 min (37 "C). After washing, cells were again layered over Ficoll and examined for the percentage of basophils by Giemsa staining. Purified basophils were cultured in RPMI 1640 medium containing 10% fetal calf serum, glutamine, and antibiotics a t 37 "C in a humidified CO, atmosphere as described (40,41). CML basophils were kept in culture for a t least 24 h before being analyzed for PG receptor expression.

The basophil (precursor) cell line KU812 was established from a patient suffering from CML (42) and kindly provided by Dr. K. Kishi. This cell line produces histamine and exhibits a cell surface mem- brane phenotype likewise expressed on normal or primary CML basophils (42, 43). KU812 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum as described for primary basophils.

Stimulation of KU812 Cells with Recombinant Human Cytokines- In order to determine the response of the basophil PG receptor/ cAMP system to rhILs, KU812 cells were incubated with various concentrations of rhIL-2, rhIL-3, or control medium for various periods of time (5 min to 24 h) in RPMI 1640 medium containing 10% fetal calf serum a t 37 "C. Thereafter, cells were washed in 50 mM Tris-HC1 buffer, pH 7.5, and analyzed for PG receptor expression and cellular levels of CAMP.

In selected experiments, PG receptor expression as well as cAMP levels were measured in KU812 cells after incubation with various concentrations of rhIL-2 or rhIL-3 for 8 h followed by short term (30 min) exposure to iloprost, PGE,, or PGD,.

Prostaglandin Receptor Binding Assays-In order to investigate PG receptors on human basophils direct binding experiments were carried out. The conditions of the assay system were essentially the same as reported earlier (44). Cells were once washed in 50 mM Tris-HC1 buffer, pH 7.5, and then suspended in 50 mM Tris-HC1 buffer, pH 7.5, containing 5 mM MgCl,, 1 mM CaCl,, and 0.1 M NaC1. About 5 X 10' cells suspended in 5 ml were used for one series of experiments.

All incubations were done in duplicate. The within assay variability amounted to 3.6 k 0.9%, the between assay variability to 7.2 & 2.0%.

In initial experiments the time course of association in binding was studied by incubating the cells (about lo5 in each tube) with [3H]PG (2.5 nM) in the absence (total binding) and presence (nonspecific binding) of unlabeled PG (100 p ~ ) , respectively, for 1-120 min. The time course of dissociation of binding was studied by addition of an excess amount of unlabeled PG (100 p M ) a t different time intervals (1-60 min) a t equilibrium.

The influence of temperature on binding was studied through equilibrium experiments carried out at 4, 15, 22, and 37 "C.

In competition experiments cells were incubated a t 4 "C for 45 min with 10 nM [3H]PGE, or [ 3 H ] i l o p r ~ ~ t or 12 nM of ["HIPGD, in the absence (total binding) and presence of increasing concentrations (10"0-10-4 M) of unlabeled agonists (nonspecific binding).

In saturation experiments, cells were incubated with increasing concentrations of [3H]PG (0.1-230 nM) in the absence (total binding) and presence of the same unlabeled PG (100 p ~ , nonspecific binding).

After incubation, the reaction mixture was diluted 1:lO with buffer and filtered through Whatman G/F B filters (Maidstone, United Kingdom) which were washed twice with buffer, dried, and taken up in scintillation fluid (Pico-Flour TM30). Radioactivity was counted in a liquid scintillation counter (LKB Wallace, 1215 Rackbeta; Turku, Finland) for 1 min. Filters retained less than 1% of total radioactivity. This amount was identical for incubations of total and nonspecific binding.

Specific binding was determined as the difference of total and nonspecific binding. In typical experiments, nonspecific binding (determined in the presence of a 100-fold excess of unlabeled PG) amounted to less than 10% of total binding (SB = T B - NSB = 100 - ( 4 0 ) *go).

Measurement of CAMP-Basal cAMP formation by KU812 cells was determined after washing the intact cells in 50 mM Tris-HC1 buffer, pH 7.5, containing 0.5 mg/ml acetylsalicylic acid and 1 mM aminophylline. lo5 cells in each tube were incubated in the absence or presence of increasing prostaglandin (PGE,, PGE2, 16,16-dimethyl- PGE,, iloprost, PGI,, 6a-carba-PGI,, ZK 110 841, PGF,,) concentrations (10"0-10-4 M) for 45 min at 20 "C. Thereafter, the cells were homogenized by means of ultraturrax and ultrasound, and the reaction was stopped by rapid centrifugation at 5000 X g for 10 min (4 "C). The supernatant contained cAMP which was determined by commercially available radioimmunoassay (Amersham International, Buckinghamshire, United Kingdom) essentially according to the manufacturer's description. Briefly, samples were reacted with cAMP and rabbit antisuccinyl-CAMP serum for 3 h a t 4 "C. The antibody-bound cAMP was thereafter extracted by a donkey anti- rabbit serum, coated onto magnetizable polymer particles. Radioac- tivity was counted in a y-counter for 1 min. The sensitivity of the assay ranged from 0.25 to 16 fmol/pl.

Histamine Release Assay-Histamine release from blood basophils of nonallergic individuals (n = 3) was determined as described previously (15) after informed consent was given. Briefly, peripheral blood cells were fractionated by incubation in 1.1% dextran 70 and 0.008 mM EDTA for 90 min a t 22 "C. Cells of the granulocyte-rich upper layer were then centrifuged (200 X g, 4 "C, 8 min) and washed twice in PIPES buffer (25 mM PIPES, 110 mM NaCl, and 5 mM KC1, pH 7.35). Granulocytes were resuspended in PIPES buffer containing 2.0 mM CaC12. Cells were adjusted to a final concentration of 2.5 X 106/ml and incubated with various concentrations of agonists in 96- multiwell plates. Basophils were incubated with PGs M ) for 5 min a t 37 "C and thereafter exposed to various concentrations of anti-IgE mAB E-124-2-8 (37 "C) for another 20 min. Thereafter, cells were centrifuged (200 X g, 4 "C) and the cell free supernatants recovered.

Histamine was measured by a commercially available radioimmunoassay (Immunotech, Marseille, France) as described previously (15). Total histamine in cell suspensions were quantified after cell lysis in distilled water. Extracellular histamine was measured in cell-free supernatants after centrifugation at 4 "C.

Data Analyses-Binding data were calculated according to Scat- chard (45) using a computer program (kindly provided by Dr. K. Neumann, Bender & Co, Vienna, Austria) which searched systemat- ically for the highest level of correlation under the model of 2 straight lines in the given interval and tested against the alternative of a single straight line approximation. This program is based on classical least squares methodology for the lines fit. The program uses a straightforward partitioning of regression sums of squares followed by a standard regression F-test. The corresponding test has been

12702 Prostaglandin Receptors on Human Basophils shown within a Monte Carlo simulation to be rather reliable and on the conservative side. The purpose of the Monte Carlo simulation was to show that the implicit multiple decision problem does not seriously affect the significance levels.

Statistical analysis was done by standard statistical tests including Student's t test and ANOVA at a confidence level of 95%.

RESULTS

Initial PG-binding Studies-In initial experiments the in- teraction of [3H]iloprost, a chemically stable PG12 analogue, and [3H]PGEl with washed KU812 cells was assessed as a function of time and temperature. Both PGs bound to the washed cells at 22 "C, and the time course of the binding reaction showed a rapid increase of binding for approximately 5 min and reached an apparent equilibrium at 15 min. In the same experiment, after a 45-min incubation time, displacement of 3H-labeled ligand by an excess of 100 p~ unlabeled agonist could be achieved, indicating that 85% of the bound material was not incorporated under these conditions. No significant difference between the binding isotherms of PGE, and iloprost (Fig. 1) was found.

The interactions were slightly temperature-dependent. At 22 "C, binding of [3H]iloprost ([3H]PGEl) at 45 min was 96% (97%) of that observed at 4 "C, at 15 "C it was 98% (97%), and at 37 "C it was 87% (91%). In all subsequent experiments, PG binding was measured at 4 "C chosing a 45-min incubation time to ensure equilibrium.

Binding of [3H]iloprost ([3H]PGE1) in the presence of an excess of 100 p h ~ agonist was less than 15% (13%) of the total binding observed in the absence of the same unlabeled agonist. Incubations of [3H]iloprost ( [3H]PGE1) prepared to different specific activities with the same unlabeled agonist with basophils, while maintaining a constant total PG concentration of 20 nM, indicated a linear relationship of basophil-bound ["HIPG to percent [3H]PG present in the incubation mixture. This reflects the same binding behavior of [3H]PG and unlabeled PG to KU812 cells.

Binding of r3H]PGs to Highly Enriched Human Blood Ba- sophils-In order to demonstrate expression of PG-binding sites on primary cells, we isolated peripheral blood basophils to near homogeneity from two CML patients. The purities of the basophil preparations as assessed by Giemsa staining were 92 and 97%, respectively. Almost all cells (>go%) were viable by dye exclusion criteria. After the isolation procedure, CML

0' 20 La 60 80 100 120

minutes

FIG. 1. Time course of specific binding of [3H]iloprost to KUSl2 cells. Association (0): [3H]iloprost (2.5 nM) was incubated with KU812 cells in the absence (total binding) and presence (nonspecific binding) of unlabeled iloprost (100 p ~ ) for the time intervals indicated. Specific binding (shown), determined as the difference of total and nonspecific binding, reaches 95% at the ligand concentration studied. Dissociation (0): in order to study the time course of displacement of specific binding (shown) an excess of unlabeled iloprost (100 p M ) was added at equilibrium (45 min). [3H]Iloprost is rapidly displaceable which indicates that only a minimal amount is incorporated under these conditions. Each point represents the mean f S.D. of 3 independent experiments with KU812 cells.

basophils were kept in culture for at least 24 h before being analyzed.

The capacity to saturate the basophil-binding sites for various PGs was assessed by incubating increasing concentrations of [3H]iloprost ([3H]PGEI, [3H]PGE2, [3H]PGD2) in the absence and presence of 100 p~ of the same unlabeled agonist. Specific binding was defined by subtraction of the binding observed in the presence from that observed in the absence of unlabeled agonist. Specific binding of both 3H-iloprost (Fig. 2) and [3H]PGEl (Fig. 3) to washed human basophils of CML patients (n = 2) was saturable and indicated a heterogeneous population of binding sites, i.e. a high affinity low capacity binding site and a low affinity high capacity binding site. [3H] Iloprost high affinity sites were capable of binding 324 f 145 fmol/lOs basophils (ie. 1,950 f 872 molecules/cell; K d = 0.5 f 0.3 nM) and low affinity sites of 2541 f 381 fmol/108 basophils (i.e. 15,297 f 2,294 molecules/celb K d = 27 f 6 nM). [3H]PGE1 high affinity sites bound 217 f 65 fmol/lO* basophils (i.e. 1,306 f 391 molecules/cell; K d = 0.5 f 0.2 nM) and low affinity sites 2,462 f 381 fmol/108 basophils (i.e. 14,821 k 2,294 molecules/cell; K d = 47 f 20 nM). At concentrations above 80 nM [3H]iloprost and 50 nM [3H]PGEl, saturation was obtained.

[3H]PGD2 was found to bind to a single class of binding site (Fig. 4) capable of binding 378 f 98 fmol/lOs cells (i.e.

3H-llopmst InMI

bound

FIG. 2. Saturation curve ( A ) and corresponding Scatchard plot ( B ) of [3H]iloprost binding to highly enriched human basophils. Basophils were enriched as described in the text and incubated with increasing concentrations of labeled [3H]iloprost. Specific binding (A) was calculated by subtracting the amount of [3H] iloprost bound in the presence of excess of unlabeled iloprost (100 p ~ ; nonspecific binding, 0) from that bound in its absence (total binding, 0). Scatchard analysis indicated heterogeneity of the binding sites with high affinity receptors capable of binding 221 fmol/108 basophils (i.e. 1,330 molecules/cell; Kd = 0.3 nM) and low affinity receptors capable of binding 2271 fmol/108 basophils (i.e. 13,671 molecules/cell; Kd = 31 nM). The results obtained with one of two patients are shown. The other patient had a high affinity binding capacity of 427 fmol/108 cells (i.e. 2,570 fmol/cell; Kd = 0.7 KIM) and a low affinity binding capacity of 2,811 fmol/108 cells (i.e. 16,922 fmOl/Cell; Kd = 23 nM).

Prostaglandin Receptors on Human Basophils

61

12703

3H-PGEl InMI

0.30 I* 0 . 2 4 B

bound

FIG. 3. Saturation curve ( A ) and corresponding Scatchard plot ( B ) or [3H]PGEI binding to highly enriched human basophils. Each assay tube contained the indicated concentrations of ["HIPGE,. Specific binding (A) was calculated by subtracting the amount of ['HIPGE, bound in the presence of excess of unlabeled PGE, (100 pM; nonspecific binding, 0) from that bound in its absence (total binding, 0). Scatchard analysis indicated heterogeneity of the binding sites with high affinity receptors capable of binding 171 fmol/ 10" basophils (i.e. 1,029 molecules/cell; Kd = 0.4 nM) and low affinity receptors capable of binding 2,192 fmol/108 basophils ( i e . 13,195 molecules/cell; Kd = 33 nM). The results obtained with one of two donors are shown. The other patient had a high affinity binding capacity of 263 fmol/108 cells (i.e. 1,583 molecules/cell; Kd = 0.7 nM) and a low affinity binding capacity of 2,732 fmol/108 cells (16,446 molecules/cell; Kd = 61 nM).

2,275 f 590 molecules/cell) with intermediate binding affinity (Kd = 13 f 4 nM) compared with the receptors (low and high affinity) binding PGEl or PG12.

Specific saturable [3H]PGE2-binding sites were not demon- strable (Fig. 5), i.e. no significant difference between nonspecific and total binding of [3H]PGE2 to basophils was found. In contrast, however, we were able to identify a high affinity PGE2 receptor site on human monocytes.2

Unlabeled iloprost and PG12 caused significant inhibition of [3H]ilopr~st binding to washed human basophils with ICso values of 11 f 3 and 11 f 1 nM, respectively (Fig. 6A). PGEl was a somewhat weaker competitor (IC50 = 16 f 2 nM), whereas PGD, (ICB0 = 950 f 70 nM) and PGE2 (ICso = 9 f 1 p ~ ) were much weaker agonists, and PGFze was ineffective (ICs0 > 100 pM). With respect to [3H]PGEl binding (Fig. 6B), a similar agonism was found with PGE, being the more effective competitor (ICB0 = 10 f 4 nM) and iloprost (IC6o = 14 f 2 nM) and PG12 (ICB0 20 f 7 nM) somewhat weaker agonists.

[3H]PGD2 binding to basophils was best displaced by PGD2 (ICB0 = 33 f 14 nM), whereas all other PGs were weaker agonists (Fig. 6C).

As expected from the almost identical chemical properties (46), no significant difference between PGIz and iloprost were found with respect to their PG receptor binding and competition behavior. Binding of PGs to KU812 Cells-The pattern of PG receptor

' I. Virgolini, S. Li, C . Sillaber, 0. Majdic, H. Sinzinger, K. Lechner, P. Bettelheim, and P. Valent, unpublished observations.

0 ' \ 50 1W I50 100 250 IW

bound

FIG. 4. Saturation curve ( A ) and corresponding Scatchard plot ( B ) of [3H]PGDz binding to highly enriched human basophils. Basophils were exposed to various concentrations of [3H]PGD2. Specific binding (A) was calculated by subtracting the amount of [3H] PGD, bound in the presence of excess of unlabeled PGD, (100 p ~ ; nonspecific binding, 0) from that bound in its absence (total binding, 0). Scatchard analysis indicated a single binding site distinct from [3H]il~prost/[3H]PGEl sites which were capable of binding 309 fmol/ 10' basophils (i.e. 1,860 molecules/cell; Kd = 16 nM). The results obtained with one of two donors are shown. The other patient had a binding capacity of 448 fmol/lO' cells ( i .e . 2,697 molecules/cell; Kd = 11 nM).

30 60 90 120

3H-PGE2 (nM)

FIG. 5. Binding of [3H]PGEz binding to highly enriched human basophils. No specific binding (determined as the difference of total binding (0) and nonspecific binding (measured in presence of 100 WM unlabeled PGE,, 0) was evident. The results obtained with one of two donors are shown.

expression [3H]iloprost, [3H]PGE1, and [3H]PGDz) on KU812 cells and isolated human blood basophils was identical. In particular, on the surface of KU812 cells two saturable binding classes, a lower capacity high affinity binding site and a higher capacity low affinity binding site were evident for both [3H] iloprost (Table I) and [3H]PGEl (Table 11), and a distinct, single "intermediate affinity" binding class for [3H]PGD2 (Table 111). As with primary blood basophils no specific saturable [3H]PGE2-binding sites could be detected for KU812 cells.

Table IV lists the relative displacement potencies for various prostaglandin agonists for [3H]iloprost, [3H]PGEl, and

Prostaglandin Receptors on Human Basophils

l ~ - ~ o 10-9 10-8 10-7 10-6 10-5 10-4

prostaglandin l M 1 R

w)-m 1 u 9 10-8 10-7 10-6 10-5 luL prostaglandin IM)

C 1M

I

a' 81 0)

e E t 3

0

I

0" a, u ._ = !i - e o ,

lo-lo 1&7 1,+6 ,d-s prostaglandin (MI

FIG. 6. Ability of unlabeled PGs to compete with [3H]PG for binding to highly enriched human basophils. Each assay tube contained 10 nM [3H]iloprost ( A ) or [3H]PGE, ( B ) , or 12 nM [3H] PGD2 (C), and the indicated concentrations of unlabeled PGs (iloprost A), PGI, (O), PGE, (VI, PGD, (O), PGEz (O), and PGF,, (0)). The 100% control value for [3H]iloprost binding ( A ) in the absence of unlabeled agonists was 567 fmol bound/1O8 cells. The ICso values were 11 c 3 nM for iloprost, 11 k 1 nM for PGIZ, 16 k 2 nM for PGE,, 950 & 70 nM for PGD,, 9 & 1 p~ for PGE,, and >lo0 p~ for PGF,,. The 100% control value for [3H]PGE, binding ( B ) in the absence of unlabeled agonists was 712 fmol bound/1O8 cells. The IC6o values were 14 f 2 nM for iloprost, 20 f 7 nM for PG12, 10 f 4 nM for PGE,, 4 f 0.7 p~ for PGD,, 17 f 4 p M for PGE,, and >lo0 p M for PGF,,. The 100% control value for [3H]PGD2 binding (C) in the absence of unlabeled agonists was 182 fmol bound/lO* cells. The ICs0 value amounted to 33 f 7 nM for PGDZ. All other PGs were much weaker competitors (ICs0 values: iloprost, 550 f 240 nM; PGI2, 900 f 140 nM. PGE,, 1 2 f 4 p ~ ; PGEz, 30 14 p ~ ; PGF,, > 100 p ~ ) . The means obtained with two donors are shown.

["H]PGD2 binding onto KU812 basophils. The rank order of potency for agonist action is very similar at PGE, and PGI, sites. However, according to previous results with nonbasophil cells (47-49), cicaprost was an effective competitor at PGI, sites but only a very weak competitor at PGEl sites. Further- more, 6a-carba-PGI, was found to compete about 4 times weaker at PGI, sites as compared to iloprost, whereas no difference at PGI, sites was found. Surprisingly, cicaprost maintained quite high activity at PGD, sites. The stable analog of [3H]PGD2, ZK 110841 (50), was the most effective substance in competing for [3H]PGD, sites. It is also note- worthy that in comparison to natural PGE,, the active metab- olite 13,14-dihydro-PGE1 was about 15 times less active at PGI, (iloprost) sites, whereas it was an even better competitor a t PGE, sites. Taken together, these findings suggest that

basophils express specific receptors for PGEI, PGI, as well as for PGD,.

Effects of Interleukin Stimulation on Binding of PGs to KU812 Cells-Incubation of KU812 cells with rhIL-2 for 8 h had no effect on [3H]PG binding, whereas incubation with rhIL-3 for 8 h dose dependently decreased the apparent number of high and low affinity binding sites without significant change in binding affinity constants (Tables I and 11). At a concentration of 1000 units/ml of rhIL-3, high affinity sites for [3H]PGEl ([3H]iloprost) were depressed by 9% (20%) of control and low affinity sites by 36% (50%) (p < 0.001).

Effects of PGs on Intracellular cAMP Formation-Various PG agonists dose dependently enhanced cAMP formation by KU812 cells (Fig. 7). Iloprost, cicaprost, PGI,, and PGE, were the most effective substances with EDso values of 2.4 f 1.6, 2.3 f 1.2, 2.8 f 2.3, and 1.7 f 1.1 nM, respectively. PGD, was about 10 times less effective with an ED50 value of 76 f 14 nM. PGEz (ED50 = 1 2 0.2 PM) and PGF,, (EDs0 >lo0 PM) were weak agonists.

Effects of Interleukins on Intracellular CAMP Formatwn- In contrast to the PGs, rhIL-3 dose dependently decreased cAMP formation by KU812 cells with an ICso value of 15 + 7 units/ml, whereas rhIL-2 had no effect (Fig. 8A). Studies on the time course (Fig. 8B) revealed that the rhIL-3-induced decrease in cAMP formation appeared rapidly after stimulation and reached an apparent equilibrium at about 30 min which remained stable for at least 8 h. Thereafter, the effect was reversible and baseline values were found at 24 h after addition of the interleukin.

Effects of PGs on rhIL-3-inhibited cAMP Formation-In a further set of experiments we addressed the question as to whether the inhibitory effect of rhIL-3 on intracellular cAMP formation would be antagonized by the PGs. After preincubation of KU812 cells with rhIL-3 for 6 h, the cells were further incubated with iloprost, PGE,, or PGD, before cAMP formation by the cells was measured. As shown in Fig. 9, the three PGs were able to interfere with rhIL-3-inhibited cAMP formation, however, PGE, and iloprost were more effective than PGD,. This effect occurred in relatively high PG concentrations (1 PM).

Effects of PGs on the Releasability of Blood Basophils of Nonallergic Donors-To confirm the biological activity of PGs, histamine release experiments were performed using granulocyte-rich cell populations obtained from nonallergic donors (n = 3). According to previous findings (17-19), PGEl

M), as well as PGE, M), reduced the capacity of the basophils to release histamine upon induction with anti- IgE. We also observed a slight inhibition of histamine releasability when basophils were pretreated with PGI, (10"j M). In contrast, PGD, M ) showed no effect, and in one of the three donors, PGD, was found to even cause a slight increase in releasability of human basophils (Table V). The effect of PGEl on basophil releasability was dose-dependent. Maxi- mum release was obtained when basophils were preincubated with M PGE,. Between 10"' and lo-' M no significant effect of PGE, was observed (data not shown).

DISCUSSION

A number of previous observations have suggested that PGs are involved in the regulation of basophil activation for mediator release (17-21, 24). Since enriched populations of basophils were found to respond to PGs, a basophil PG receptor has been postulated ( 5 ) . However, the cellular substrate remained to be presented. By the use of a radioreceptor assay we were now able to characterize PG-binding sites expressed on human basophils. On the cell surface of highly enriched


TABLE I Effect of rhIL-2 and rhIL-3 on ['Hliloprost binding to KU812 cells (n = 6)

High affinity sites Low affinity sites

Bmax K d Bmax K d

fmolfloR cells nM /mol/loH cells nM

(728 f 192) (12,936 f 1,950)

(656 f 144) (12,292 t 3,663)

(608 f 313) (11,925 f 4,045)

(535 f 246) (10,727 f 2,751)

(313 f 115) (9,005 f 8,879)

(144 f 138) (6,514 -t 1,938)

Control 121 f 32 0.3 f 0.2 2,149 f 324 53 f 14

rhIL-2 stimulated 100 units/ml 109 f 24 0.3 f 0.17 2,042 f 592 87 -t 28

rhIL-2 stimulated (1000 units/ml) 101 f 52 0.5 f 0.3 1,981 f 672 77 f 21

rhIL-3 stimulated (10 units/ml) 89 f 41 0.6 f 0.4 1,782 f 457 72 + 19

rhIL-3 stimulated (100 units/ml) 52 f 19" 0.4 f 0.3 1,496 t 384" 79 f 17

rhIL-3 stimulated (1000 units/ml) 24 f 23' 0.5 f 0.6 1,082 f 322' 74 f 23

Values in parentheses: estimated apparent sites/cell, p < 0.01. ANOVA versus control. Values in parentheses: estimated apparent sites/cell, p < 0.001. ANOVA uersus control.

TABLE I1 Effect of rhIL-2 and rhIL-3 on ['HIPGE, binding to KU812 cells (n = 31

High affinity sites" Low affinity sites

Bmax K d Emax K d

fmol/loR cells nM fmol/loR cells nM Control 209 f 27 0.5 f 0.2 2,451 f 309 49 t 8

rhIL-2 stimulated 100 units/ml 189 f 31 0.4 f 0.2 2,349 f 676 67 f 19

rhIL-2 stimulated (500 units/ml) 178 f 47 0.5 f 0.5 2,089 f 561 63 f 24

rhIL-3 stimulated (10 units/ml) 76 f 53" 0.7 f 0.5 1,656 f 552 54 f 21

rhIL-3 stimulated (100 units/ml) 54 f 23' 0.6 f 0.4 1,254 f 457" 65 + 27

rhIL-3 stimulated (1000 units/ml) 19 f 21' 0.6 f 0.6 889 t 422' 77 f 23

(1,258 f 102) (14,755 f 1,860)

(3,949 f 867) (14,140 f 4,069)

(1,071 f 282) (12,575 -t 3,377)

(457 f 319) (9,969 f 3,323)

(325 t 138) (7,549 f 2,751)

(114 f 126) (5,351 f 2,540) ' Values in parentheses: estimated apparent sites/cell, p < 0.05. ANOVA versus control. I, Values in parentheses: estimated apparent sites/cell, p < 0.01. ANOVA versus control. Values in parentheses: estimated apparent sites/cell, p < 0.001. ANOVA versus control.

TABLE I11 Effect of rhIL-2 and rhIL-3 on L3H]PGD2 binding to

KU812 cells (n = 3) Bmax K d

/rnol/10" cells Control 404 f 35

nM

(2,432 f 210) 13 + 4

rhIL-2 stimulated Not tested (100 units/ml)

rhIL-2 stimulated 456 f 43 (1000 units/ml)

12 f 8 (2,745 f 259)


rhIL-3 stimulated 294 f 53" 15 f 7 (100 units/ml) (1,769 f 319)


Values in parentheses: estimated apparent sites/cell, p < 0.05. ANOVA versus control.

CML basophils, as well as on the basophil cell line KU812, we were able to show specific binding for [3H]PGEI, ['HI iloprost, and [3H]PGD2 and identified two classes of PGE1- binding sites, two classes of PGIz-binding sites, and a separate single class of PGDp-binding sites. Specific saturable cell surface receptors for PGE, and PGF,, could not be detected. The capacity of CML basophils, as well as KU812 cells, to bind the PGs and the affinity constants of the basophil PG receptors were found to correspond in approximation to the

TABLE IV Relative displacement potencies (IC5,,) of PG binding to PG receptors

expressed on KU812 cells n > 3 in duplicate incubation series each; ic f S.D.

["HIIloprost ['HH]PGE1 ['H]PGDp PGEZ 10 f 2 nM 13 f 2 nM 190 k 45 nM Iloprost 8 f 4 n ~ 1 2 f l n M 1 5 0 f 3 0 n ~ Cicaprost 6a-Carba-PG12

9 + 3 n M 6 f 2 p M 1 2 0 f 5 0 n ~ 12 k 5 nM 50 f 5 nM 300 f 100 nM

13,14-Dihydro-PGE, 100 f 21 nM 6 t 1 nM 2.8 f 0.4 p M 7 + 2 p M 1 4 + 3 p M 2 5 f 5 p M

16,16-Dimethyl-PGE2 3 f 4 p M 7 f 3 p M 5 f 0.8 p M

ZK 110 841 1 t 0.2 p M 2 f 0.5 p M 22 f 8 nM

0.9 f 0.2 p M 1 f 0.3 p M 15 f 4 nM PGFz,, >loo p M >loo p M >loo p M

PGE, 1 2 f 5 n ~ 1 1 f 2 n M 3 f 0 . 5 p M

PGE,

PGDZ

binding data obtained for other leukocytes (29-31). No substantial differences in receptor density, binding affinity constants, or distribution of PG receptors between primary CML basophils and the KU812 cell line were found. The exact number and binding characteristics of PG receptors expressed on normal human basophils remain unknown, since it is virtually impossible to enrich larger numbers of normal human blood basophils to homogeneity for binding experiments.

The observation that CML basophils, as well as KU812 cells, express IP (PG12) receptors is a novel finding. In order to demonstrate binding of PGI, to basophils we have used the

12706 Prostaglandin Receptors on Human Basophils

,LY L 1 , . , , , . ,..,,.., . . . ,1111 ,,,,..." . / , , , eo X r 9 lo" 10' 10-6 lb5 10"

prostaglandin IM)

FIG. 7. Effect of PGS on intracellular cAMP formation (radioimmunoassay). Intracellular cAMP formation by KU812 cells was measured after a 45-min incubation with various PG concentrations at 37 "C. The corresponding EDm values were 1.7 f 1.1 nM for PGE, (V), 2.4 f 1.6 nM for iloprost (A), 2.8 f 2.3 nM for PG12 (o), 3.1 f 1.9 nM for cicaprost (+), 10 f 2 nM for ZK 110 841 (U), 76 f 14 nM for PGD, (O), 0.9 +- 0.1 PM for 16,16-dimethyl-PGE2 (X), 1 f 0.2 p M for PGE, (0), and >lo0 p M for PGF,, (0). The means of six independent experiments with KU812 cells are shown. The baseline value amounted to 2,088 f 312 fmol cAMP/108 KU812 cells.

A t 120

1 XI 1w ma interleukin l U / m l l

2 L 6 U 10 12 I' 16 18 20 1 2 24

hours

FIG. 8. Effect of interleukins on intracellular cAMP formation (radioimmunoassay). KU812 cells were incubated with rhIL-2 (V) and rhIL-3 (0) (100 units/ml each) at 37 "C for 6 h. cAMP was measured as described under "Experimental Procedures." The baseline value amounted to 2,088 f 312 fmol cAMP/108 KU812 cells. A, whereas rhIL-2 had no significant effect on cAMP formation by KU812 cells, cAMP formation was dose dependently inhibited by rhIL-3 (IC6" 15 f 7 units/ml). B, cAMP inhibition by rhIL-3 was time-dependent reaching equilibration at about 30 min. cAMP levels slowly recovered during the following 24 h.

chemically stable and biologically active PGIz mimetic [''Hliloprost as the principle radioligand (46). This compound has repeatedly been chosen to detect and characterize PG12- binding sites on various mesenchymal including circulating blood cells (47-49,51,52). Since it is well known that iloprost (as well as natural PG12) exhibits substantial affinity for EP receptors (53, 54), we used several unlabeled IP agonists, including cicaprost, an IP receptor-specific compound. As expected from previous findings with nonbasophil cells (47- 49), the IP agonists were found to be potent competitor ligands at PG receptors expressed on human basophils. How-

C I 200 %

150

100

50

0

FIG. 9. Effect of PGs on rhIL-3-induced decrease in cAMP levels in KU812 cells. KU812 cells were incubated with rhIL-2 (0) and rhIL-3 (0) (100 units/ml) at 37 "C for 6 h, washed, and then incubated with PGE,. ( A ) , iloprost ( B ) , or PGD, (C). cAMP was measured as described under "Experimental Procedures." The baseline value amounted to 2,088 f 312 fmol cAMP/108 cells in controls and to 1,239 f 351 fmol cAMP/108 cells after stimulation with rhIL- 3. Control, without IL is indicated by 0.

TABLE V PG M) induced changes in basophil releasability

M) for 5 min, and then challenged with various concentrations (see text) of mAb E-124-2-8 (anti-IgE) for 20 min. Thereafter, cells were centrifuged at 4 "C and the cell-free supernatant analyzed for the presence of histamine. Results represent PG-induced changes (percent of control) in histamine release obtained at the optimal concentration (inducing the maximum histamine release) of anti-IgE. The release obtained with control medium (=spontaneous release) was less than 5% in all exDeriments.

Isolated human blood granulocytes were incubated with PGs

Preincubation stimulus

IgE-mediated histamine release

Donor 1 Donor 2 Donor 3 % of control

Control 100 100 100 PGE, 72 55 Not tested PGEZ 65 57 85 PGD, 98 105 125 PGI2 89 76 Not tested

ever, whereas natural PG12, iloprost, and 6a-carba-PGIz were found to be effective in competing for binding with [3H] iloprost, as well as [3H]PGEl to the basophils, cicaprost was only a potent competitor for binding to PGL, but not to PGEl receptors. Taken together, these results suggest that human basophils express, in addition to PGE,-binding sites, specific PG12 receptors.

According to previous observations, human basophils were


found to express functionally active PGEl-binding sites. By using natural [3H]PGEl as radioligand we identified two classes of saturable PGEl-binding sites on human basophils. Recently, evidence for heterogeneity of PGEl-binding sites has emerged (for review, see Ref. 55). One subtype of PGE1 receptor binds PGI, with substantial affinity, whereas another subtype also binds PGE,. Our results suggest that PGI, (but not cicaprost) binds with high affinity to a PGEl receptor expressed on human basophils. Whether or not these basophil “PGEl/PG12 receptors” differ from those expressed by other cells such as the neuronal hybrid cell line NCB-20 (56), the mastocytoma cell line P-815 (57), human platelets (58-61), macrophages (30), or MEG-01 cells (51) (probably due to species and/or tissue heterogeneities, see Refs. 62 and 63) remains unknown. Also whether the basophil receptors for PGE, and the PGI, receptor are related in molecular terms (as has been suggested for human platelets (59)) remains to be determined. PGE, was also found to bind to the basophil PGE, receptor. However, in contrast to the above mentioned cells no binding site equally specific for PGEl and PGEz could be demonstrated on human basophils. Thus, the possibility that this PGE, receptor is related to the platelet receptor for PGE, (which also binds PGEz with substantial affinity) seems rather unlikely. Furthermore, by the technique applied in this study no specific saturable PGE, receptors could be identified on human basophils.

PGE, receptors and respective subtypes (EP-1, EP-2, and EP-3 receptors) have recently been identified (49, 53, 54). The observation that human basophils do not express specific saturable PGE, receptors was an unexpected finding since a response of human basophils (and of KU812 cells) to PGE, was observed by us and by others (17-21, 24). This might be due to an activation through another PG-binding site such as the PGE, or PGI, receptors which also bind PGE,. It can be deduced from Fig. 6 and Table IV that PGE, maintains weak agonist activity at PGI,, PGE,, as well as PGD2 receptors. 16,16-Dimethyl-PGE2, although being a weak agonist, was about two times more potent as compared to natural PGE, in displacing [ 3 H ] i l ~ p r ~ ~ t , [3H]PGEI, and [3H]PGD2 from their specific binding to KU812 cells. Furthermore, a tendency for biphasic PGE, competition curves was found at PGE, receptors (Fig. 6B) resolving in pseudo-Hill plots below unity (not shown) that may also imply displacement from two different components which may represent a PGE, receptor (with higher affinity) as well as a PGI, receptor (with lower affinity) (49). Another possibility could be that basophils express extremely low numbers of saturable PGE, receptors, or specific receptors with extremely low affinity for PGE, which could not be detected by the receptor assay used. Some of the effects of PGE, could also be mediated via accessory cells involving a paracrine route.

The specific binding of [3H]PGD2 to human basophils, as well as KU812 cells appeared homogeneous resolving in straight Scatchard plots with linear regression coefficients that ranged between -0.93 and 0.98%. The observation that human blood basophils express PGD2-binding sites is a novel finding, although the possibility has already been suggested by MacGlashan et al. (5). Similar to his results we observed no decrease in the releasability of human blood basophils upon induction with PGD,. This is of particular interest since the other PGs (PGE1, PGE,, and PGI,). were found to decrease the releasability in the basophils. Furthermore, unlike PGEl and PGI,, PGD, failed to counteract the rhIL-3-induced inhibition of cAMP formation in KU812 cells. The PGD, receptor differs from the other PG receptors expressed by basophils in several aspects. In contrast to the other PG-

binding sites, no high capacity low affinity binding component of the PGD, receptor could be demonstrated, and the binding affinity for the natural ligand was intermediate ( K d 10-20 nM) as compared to the affinities (low and high) of PGE1- and PGI,-binding sites for their natural ligands. This, as well as the much weaker potency in stimulating cAMP formation corresponds to PGD, receptors expressed on human platelets (60), although the ICbo values for iloprost and PGI, competition at PGD, receptors were higher for primary human basophils as compared to human platelets (data not given). In fact, only natural PGD, (22 f 8 nM) and its orally active mimetic ZK 110 841 (50), which was an even better competitor (5 f 0.8 nM), were potent agonists at the [3H]PGDz sites.

Little is so far known about the half-life, degradation, catabolism, and regulation of PG receptors on human basophils. The PG receptors on human basophils and KU812 cells seem to be expressed in a constitutive manner. We observed a rather stable configuration of bound PGs to KU812 cells after a 45-min observation period without measurable inter- nalization or degradation. In a set of experiments we tried to identify conditions which modulate expression of PG-binding sites. For this purpose we analyzed PG receptor expression after induction with two cytokines for which basophils are known to express cell surface receptors. IL-3 is a pleiotropic cytokine and has recently been shown to activate human basophils for mediator secretion via high affinity binding sites (15). IL-2 binds to basophils and KU812 cells through low affinity binding sites (65) and has no effect on basophil releasability. We observed down-regulation of PGEl and PGIZ-binding sites upon stimulation with rhIL-3 but not rhIL- 2. Furthermore, a significant, dose-dependent decrease in cAMP formation by KU812 cells upon induction with rhIL-3 was found. Maximum down-regulation upon induction with rhIL-3 was observed after 30 min. Whether the rhIL-3-induced changes in the releasability of human basophils are associated with or even due to changes in expression of cAMP and/or PG receptors remains unknown. IL-2 showed no effect on cAMP levels or PG receptor expression on human basophils.

From a number of previous (24) and more recent studies (5), as well as from the above data, it might be deduced that the changes in the levels of cellular cAMP are inversely related to the capacity of the human basophils to release mediator molecules. In this study, all PGs tested induced an increase in cAMP levels in KU812 cells with PGEl and PG12 being the most potent agonists, whereas rhIL-3 was found to induce a significant decrease in cAMP levels. In addition, PGEl and PGIz (but not PGD,) were able to counteract the effect of rhIL-3 on cAMP levels in KU812 cells. This would be in agreement with the above mentioned hypothesis. On the other hand, the effects of PGD, in this respect are difficult to explain since PGD, failed to down-regulate releasability in normal human blood basophils but up-regulates cAMP levels in KU812 cells. In our study this discrepancy may well be due to the different cell types analyzed. However, a similar discrepancy concerning PGD, effects has been described by Holgate et al. (66) for rat serosal mast cells. He suggested an action of PGD, on the noninhibitory intracellular pool of cyclic AMP. Thus, the change in cAMP may not necessarily predict or determine a particular response of the secretory machinery in basophils or mast cells.

In general, it is assumed that the concentration of an agonist exhibiting its biologic activity would be in the same range as the association constant of its cell surface receptor. If such a concept applies to the basophil PG receptors, the concentration range of PGE, or PGI, required to modulate

12708 Prostaglandin Receptol

cellular functions suggest that the lower affinity high capacity binding sites may play a more important role in signal trans- duction (associated with basophil releasability) compared to the higher affinity low capacity binding sites. If so, this might explain the failure of the PGDz receptor (lacking a low affinity high capacity binding class) to transmit a release (down) regulating signal similar to the PGEl and PGIz receptor(s).

On the basis of agonist potency, PG receptors have been subclassified (55). For example, there is now good evidence for distinct IP and EP receptors (EP1, EPz, and EP3 subtypes), and the PG receptor subtypes may mediate different cellular responses (53, 54). Furthermore, PG receptors may bind two (or more) different PGs with equal affinity (PGEl and PG12, or PGEl and PGEz) (51). However, an exact classification of PG receptor subtypes will not become available unless the respective subtrates (proteins) have been cloned and se- quenced.

So far, PG receptors have been detected on various leukocytes (29-31) and platelets (46, 51, 58-61), as well as on a plenty of tissue-fixed cells (44, 47-49, 52, 62, 63). We here provide evidence that human basophils express PG-binding sites. The basophil receptors for PGEl and PGIz may play an important role in the regulation of basophil activation and mediator secretion during allergic responses.

Acknowledgment-We thank Eva Spanblochl for skillful technical assistance.

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