©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epitope-labeled Soluble Human Interleukin-5 (IL-5) Receptors
AFFINITY CROSS-LINK LABELING, IL-5 BINDING, AND BIOLOGICAL ACTIVITY (*)

(Received for publication, April 3, 1995; and in revised form, September 6, 1995)

Pamela M. Brown Philip Tagari Kevin R. Rowan Violeta L. Yu Gary P. O'Neill C. Russell Middaugh (1) Gautam Sanyal (1) Anthony W. Ford-Hutchinson Donald W. Nicholson (§)

From the Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P. O. Box 1005, Pointe Claire-Dorval, Quebec H9R 4P8, Canada Department of Pharmaceutical Research, Merck & Co., West Point, Pennsylvania 19486

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human receptor for the potent eosinophilopoietic cytokine interleukin-5 (IL-5) consists of two components: a 60-kDa ligand-binding alpha chain (IL-5alphaR) and a 130-kDa beta chain (IL-5betaR). Three ectodomain constructs of the alpha chain (alphaRED) bearing C-terminal epitope tags were engineered and expressed in baculovirus-infected Sf9 cells. Each recombinant alpha chain was secreted into the medium, maximum expression occurring 72 h post-infection. The various soluble alpha chains were shown by affinity cross-link labeling and competition with unlabeled IL-5 to bind recombinant human (rh) I-IL-5 specifically with an ED of 2-5 nM. The epitope tag provided a simple purification of the receptor from conditioned medium using immunoaffinity chromatography. The purified material had an apparent molecular mass of 43 kDa and was heterogeneously glycosylated. Sedimentation analysis revealed a 1:1 association of the purified epitope-tagged soluble receptor with its ligand, resulting in the formation of a 70-74-kDa complex. Circular dichroism analysis revealed that the soluble alpha chain existed with a significantly ordered structure consisting of 42% beta-sheet and 6% alpha-helix. Such analyses combined with fluorescence spectrometry suggested that ligand-receptor complex formation in solution resulted in minimal conformational changes, consistent with the suggestion that the membrane-associated form of the alpha chain itself has minimal signal transduction capability. Surface plasmon resonance studies of the interaction of the purified alphaRED with immobilized rhIL-5 revealed a specific, competable interaction with a dissociation constant of 9 nM. Preincubation of an IL-5-dependent cell line with the epitope-tagged alphaRED also dose-dependently neutralized rhIL-5-induced proliferation. These data demonstrate that biologically active epitope-tagged recombinant soluble IL-5 receptors are facile to produce in large quantities and may have therapeutic utility in the modulation of IL-5-dependent eosinophilia in man.


INTRODUCTION

Interleukin-5 (IL-5) (^1)is a pleiotropic cytokine with demonstrated activatory and proliferative effects on murine B cells, T cells, and eosinophils. In humans, IL-5 activity appears to be restricted to eosinophil/basophil lineages and may play a role in the regulation of eosinophilic inflammation associated with various diseases, notably bronchial asthma(1) .

The human receptor for IL-5 consists of two components: a 60-kDa ligand-binding alpha chain (IL-5alphaR) and a 130-kDa beta chain (IL-5betaR), which does not by itself bind IL-5 but forms a high affinity signal transducing receptor with the IL-5/alpha chain complex(2) . The cloning and characterization of the IL-5 receptor alpha chain identified two mRNA species that correspond to alternatively spliced soluble isoforms of the full-length transmembrane alpha chain(2, 3) . One of these alternatively spliced mRNA species is the major transcript expressed in eosinophilic HL-60 cells (4) and in eosinophils grown from cord blood(2) . These soluble receptors may play a role in the immunoregulation of eosinophilia by neutralizing the biological activity of secreted IL-5.

This report describes the baculovirus expression of three ectodomains of the ligand-binding IL-5 receptor alpha chain (S1-, S2- and S3-alphaRED). Epitope tags were engineered onto the C termini of the various constructs allowing purification in a mild single-step procedure. The purified proteins were then shown to bind IL-5 and to neutralize the biological activity of this potent pro-inflammatory hematopoietin in a cellular proliferation assay, suggesting that such recombinant soluble receptor forms may have utility in the therapy of eosinophilic inflammation.


EXPERIMENTAL PROCEDURES

Materials

The human promyelocytic leukemia cell line HL-60 (ATCC CCL 240) and murine B cell leukemia cell line BCL-1 clone 5(B)1b (ATCC TIB 197) were obtained from the American Type Culture Collection (Rockville, MD). The eosinophilic strain of HL-60 cells (HL-60/MF211#7) was developed essentially as described previously (4, 5) at the Merck Frosst Centre for Therapeutic Research (Montreal, PQ). Recombinant human interleukin-5 was purchased from R& Systems (Minneapolis, MN) or was produced in a baculovirus expression system, purified, and characterized as described elsewhere (6) . Spodoptera frugiperda (Sf9) insect cells, Autographa californica nuclear polyhedrosis virus (AcMNPV), the TA cloning kit, and a linear AcMNPV DNA transfection module were purchased from Invitrogen (San Diego, CA). The baculovirus transfer vector pETL was obtained from Dr. C. Richardson (Biotechnology Research Institute, National Research Council Canada, Montreal, PQ). The FLAG M2 monoclonal antibody and anti-FLAG M2 affinity gel were purchased from IBI Inc. (New Haven, CT). The FLAG peptide Ac-DYKDDDDK was synthesized by AnaSpec Inc. (San Jose, CA). FPLC chromatography columns were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden). Chemiluminescence Western blotting reagents, protein molecular weight markers, streptavidin-horseradish peroxidase conjugate, and sheep anti-mouse horseradish-peroxidase conjugate were from Amersham Corp. Carrier-free NI was obtained from Nordion International (Ottawa, Ontario, Cananda). The BIAcore system, CM5 sensor chips and amine coupling kit were obtained from Pharmacia Biosensor AB (Uppsala, Sweden). IODO-BEADS, bis(sulfosuccinimidyl)suberate (BS^3), and the BCA protein assay reagent kit were from Pierce. N-Glycosidase F (EC 3.2.2.18) and molecular biology reagents were from Boehringer Mannheim unless specified otherwise. Cell culture reagents were from Life Technologies, Inc., and protease inhibitors were from Sigma.

Cloning of the IL-5 Receptor alpha Chain

Total RNA was isolated from the human promyelocytic leukemia HL-60 cell line by the guanidinium isothiocyanate method(7) , and poly(A) RNA was purified by oligo(dT)-cellulose chromatography (mRNA separator kit, Clontech). The cDNA encoding the full-length IL-5 receptor alpha chain was obtained as two overlapping fragments by reverse transcription, followed by polymerase chain reaction amplification. Poly(A) HL-60 RNA (2 µg/reaction) was reverse-transcribed (10 min for 23 °C; 15 min for 42 °C; GeneAmp RNA PCR kit, Perkin-Elmer) and then heated to 95 °C for 5 min. A PCR gem was added (Ampliwax(TM) PCR gem; Perkin-Elmer) and then PCR amplification of the cDNA was performed using standard methods (3 mM MgCl(2), 2.5 units of Taq polymerase/100 µl of reaction volume; 8). The cycling conditions were as follows: 32 cycles of 94 °C, 90 s; 51 °C, 30 s; 72 °C, 90 s, followed by a 10-min extension at 72 °C.

The oligonucleotides GO78 (ATGATCATCGTGGCGCATGTATTA) and GO98 (CTACTTACCCACATAAATAGGTTGGCTCCACTCACT) were used to amplify a 1002-base pair fragment corresponding to clone lL5R.25(3) . A second pair of oligonucleotides, GO80 (CCTCCACTGAATGTCACAGCAGAG) and GO79 (TCAAAACACAGAATCCTCCAGGGT) amplified a 542-base pair fragment spanning nucleotides 721-1263 of the published IL-5 receptor alpha chain sequence (clone lh5R.12; 3). Amplified bands of the appropriate size were electrophoretically transferred to DEAE-cellulose (NA-45 DEAE-cellulose, Schleicher & Schuell, Keene, N.H.) and then directly cloned into EcoRV-digested, T-modified pBluescript II SK (Stratagene Cloning Systems, La Jolla, CA). Escherichia coli XL-1 cells were transformed, restriction digests performed on purified plasmid preparations, and insert-containing preparations sequenced using the dideoxy chain termination method (Sequenase, U. S. Biochemical Corp., Cleveland, OH) according to standard procedures(10) . The resulting plasmids containing the 5`-1002 base pairs alpha chain (clone pBSK-IL5R-alpha3) and the 3`-542 base pairs of the receptor (clone pBSK-IL-5R-alpha11) were then used to reconstruct the full-length IL-5 receptor alpha chain by restriction digestion of both pBSK-IL-5R-alpha3 and pBSK-IL-5R-alpha11 with BamHI and SacI and ligation into BamHI-digested pBSK to generate pBSK-IL-5R-alpha.

Engineering of Epitope-labeled Soluble IL-5 Receptor alpha Chains

The full-length IL-5 receptor alpha chain clone (pBSK-IL-5R-alpha) was used as a template to engineer three alternate forms of the soluble extracellular domain of the IL-5 receptor alpha chain in two sequential PCR amplification reactions as follows: the oligonucleotide DN265 (CGCGGATCCCCGCCATGATCATCGTGGCG) served as the sense primer for all three soluble forms of the IL-5 receptor alpha chain. The oligonucleotides PB20 (TCTTGAGAACCCCACATAAATAGGTTGGCTCCACTCACTCCA),PB23 (CTTACCCACATAAATAGGTTGGCTCCACTCACTCCA), andPB26 (CCACTCTCTCAAGGGCTTGTGTTCATCATTTCC) served as the antisense primers in the first round of PCR reactions to amplify the regions of cDNA corresponding to the regions encoding the various C-terminal amino acids of the three soluble receptor isoforms. Oligonucleotides PB22 (GGATCCCTATTATTTATCATCATCATCTTTATAATCTCTTGAGAACCCCACATA), PB25 (GGATCCCTATTATTTATCATCATCATCTTTATAATCCTTACCCACATAAATAGG), and PB27 (GGATCCCTATTATTTATCATCATCATCTTTATAATCCCACTCTCTCAAGGGCTT) were the antisense primers for the second round of PCR in which the 8-amino acid FLAG epitope tag followed by two stop codons and a BamHI restriction site were engineered onto the 3` end of the products of the first round of PCR. The polymerase chain reactions were carried out as described earlier in the presence of 3 mM MgCl(2), 2.5 units of Taq DNA polymerase/reaction, and cycling as follows: 20 cycles of 94 °C, 30 s; 45 °C, 30 s; 72 °C, 1 min. Amplified fragments of the appropriate size were purified and then cloned into the pCR II vector with the TA cloning kit (Invitrogen). Insert-containing clones were sequenced, and when required, restriction digestion and ligation were performed in order to construct a complete cDNA sequence free of PCR errors.

Construction of Recombinant Baculovirus

Recombinant transfer vectors pETL-alphaRED S1-, S2- and S3-FLAG were constructed by ligating the corresponding BamHI fragments into the BamHI site of pETL downstream of the polyhedrin promoter. The orientation of the RED S1-, S2-, and S3-FLAG inserts were determined by restriction mapping and the junctions confirmed by sequencing. Recombinant baculovirus was produced by in vivo homologous recombination following cationic liposome-mediated transfection of the recombinant transfer vectors in the presence on linearized AcMNPV DNA (Invitrogen) and was identified and purified according to standard procedures(11) .

Production and Purification of Soluble Recombinant alpha Chain

Insect cells (Sf9) were maintained in 1-liter suspension cultures in Grace's insect cell medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum at a cell density between 0.4 and 2.0 times 10^6 cells/ml. Infection of the insect cells was carried out under low serum conditions by first harvesting cells in log-phase growth by centrifugation at 300 times g (10 min, 22 °C), discarding the supernatant, washing the cells with serum-free medium (Sf900 II, Life Technologies, Inc.) and then centrifugation at 300 times g (10 min, 22 °C). The supernatant was discarded and the cells resuspended in Sf900 II medium to a cell density of 2-3 times 10^6 cells/ml. Recombinant baculovirus was then added at a multiplicity of infection of approximately 3. The recombinant virus-infected insect cells were harvested at 72 h postinfection (>90% trypan blue excluding) by centrifugation at 300 g. Protease inhibitors were added to the cell pellet (20 µg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor, and 34 mM E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanido)butane)), and lysis was performed in D-PBS, 1% (v/v) Triton X-100 (30 min, 22 °C) at one-tenth the original cell culture volume (20-30 times 10^6 cells/ml) of Triton lysis buffer, and cellular debris was pelleted by centrifugation (12000 times g, 5 min). Protease inhibitors were added to the supernatant (2 µg/ml each of aprotinin, leupeptin and soybean trypsin inhibitor, and 3.4 mM E-64) as was 25 mM HEPES, pH 7.4. The conditioned medium was then titrated to pH 7.4, filtered through a 0.2-µm membrane and then applied to an 8 cm times 1-cm FLAG-M2 affinity column (IBI). The column was then washed extensively with D-PBS (calcium and magnesium-free, Life Technologies, Inc.) and the alphaRED eluted with either 100 mM glycine, pH 3.0, into tubes containing one-tenth volume of 1 M Tris, pH 8.0, or with a step gradient of FLAG peptide (50 µg/ml, 100 µg/ml in D-PBS). The eluted fractions containing alphaRED were then pooled and dialyzed extensively against D-PBS. Protein concentrations were determined using the BCA protein assay reagent (Pierce) and purity of the affinity column-eluted material was determined by silver staining of SDS-polyacrylamide gels (Bio-Rad).

Immunoblot Analysis of alphaRED-FLAG

Immunoblot analysis was carried out essentially as described previously (12) following SDS-polyacrylamide gel electrophoresis under reducing conditions(13) . The alphaRED was detected following incubation with 20 µg/ml M2 FLAG monoclonal antibody (IBI) with the ECL Western blotting system (Amersham). Affinity-eluted fractions containing soluble receptor were identified by dotting small aliquots of each fraction onto nitrocellulose and then carrying out ECL immunoblot detection.

Deglycosylation

Purified alphaRED was deglycosylated with N-glycosidase F (Boehringer Mannheim, EC 3.22.18) as described previously (14) by first reducing and denaturing the alphaRED by boiling in the presence of 0.1% (w/v) SDS and 1% (v/v) beta-mercaptoethanol. 1% (w/v) n-octyl glucoside was added to the protein samples to stabilize the enzyme against inactivation by SDS, and then 2 units of N-glycosidase F were added. After a 16-h incubation at 22 °C, another 2 units of N-glycosidase were added, the samples incubated an additional 2 h at 22 °C and then subjected to SDS-PAGE and immunoblot analysis as described above.

Affinity Cross-link Labeling of alphaRED

Conditioned medium from recombinant baculovirus-infected insect cells was diluted 50-fold into D-PBS, pH 7.4, and was incubated with 0.5 nMI-rhIL-5 in the presence of 0-200 nM unlabeled rhIL-5 for 3 h at 4 °C. BS^3 was then added to a final concentration of 1 mM and incubated on ice for 10 min before quenching with 100 mM glycine pH 9.0. The cross-linked samples were then reduced and denatured (5% (v/v) beta-mercaptoethanol, 1% (w/v) SDS), subjected to SDS-PAGE, and visualized by autoradiography (Kodak X-Omat).

Nonequilibrium Binding of IL-5 to alphaRED

The kinetics of association and dissociation of IL-5 to the purified soluble receptor (alphaRED) were studied in real time, without labeling any of the reagents, using a surface plasmon resonance detector (BIAcore, Pharmacia Biosensor AB). The detector utilized in the BIAcore system monitors the refractive index at the surface of a sensor chip, which changes as the amount of adsorbed mass varies due to specific interaction between the sensor and a component of the analyte flowing across the sensor surface. The changing refractive index upon interaction was expressed in arbitrary response units (R, with R(max) designating the maximum binding capacity of the sensor surface as detailed previously; (15, 16, 17) ).

The sensor surface was prepared by covalent coupling of recombinant human IL-5 (carrier-free, R& Systems; 10 µg/ml in 20 mM NaOAc, pH 4.5) at a flow rate of 5 µl/min using the amine coupling kit containing N-hydroxysuccinimide, N-ethyl-N`-(3-diethylaminopropyl)carbodiimide hydrochloride and 1 M ethanolamine hydrochloride, pH 8.5, as described by the manufacturer (Pharmacia Biosensor AB). The extent of coupling was controlled by varying the time of sensor chip activation, IL-5 exposure, and ethanolamine quenching from 1 min/step to 7 min/step.

Sensorgrams were generated by passing alphaRED-containing analyte solutions across the IL-5-modified sensor surface from which the rate of association (k) could be derived. Dissociation rate constants (k) were similarly derived by then passing D-PBS across the IL-5 sensor/alphaRED complex surface according to the following reaction for a simple bimolecular interaction.

On-line formulae not verified for accuracy

Since IL-5 was immobilized to the sensor chip, the increase in R upon alphaRED binding was a reflection of the amount of IL-5/alphaRED complex formed and the maximum amount of IL-5/alphaRED complex that could form was determined by the amount of active IL-5 present and accessible for interaction with the analyte (R(max)).

On-line formulae not verified for accuracy

The association rate constant was derived from analysis of the association phase of sensorgrams obtained by passing varying concentrations of analyte (alphaRED) solutions across a single IL-5 sensor surface and plotting the changing response with time (dR/dt) against R at that time so that a line with slope of -(k[alphaRED] + k) or -k(s), results. When k(s) is then plotted against the concentration of alphaRED in each of the solutions the slope of the resulting line is equal to the association rate constant (k(a) or k).

The dissociation rate constant was derived from the portion of the sensorgram where the analyte solution was replaced with buffer and the dissociation of alphaRED from the IL-5 sensor surface monitored as a decrease in the response units. Integration of the first order rate equation dR/dt = kRt and plotting the initial phase of the dissociation sensorgram as ln (Rt(1)/Rt(n)) versus time resulted in a line with a slope equal to the negative of the dissociation rate constant (-k(d) or -k). The equilibrium dissociation constant (K(d)) was obtained from the equation K(d) = k/k. The IL-5 sensor surface was regenerated with a 5-µl pulse of 20 mM HCl between exposure to successive alphaRED-containing analyte solutions.

Circular Dichroism

Circular dichroism spectra were measured with a JASCO J-720 spectropolarimeter. All spectra were obtained at a resolution of 0.5 nm and bandwidth of 2 nm. In all cases, 5 spectra were averaged, a buffer base line subtracted, and final spectra smoothed by a digital filtering procedure. Protein concentrations of 1.15 or 2.3 M were used in 0.1-cm path length cells, which were thermojacketed at 22 °C. For secondary structure estimation, data were converted to mean residue ellipticity(18) . Thermal unfolding experiments were performed under the same conditions with an AVIV 62DS spectropolarimeter. Ellipticity was monitored at 5 wavelengths (208, 215, 222, 230, and 350 nm) at 2 °C increments with a 2-min incubation period at each wavelength, a period sufficient for equilibrium to be obtained.

Fluorescence Emission

Fluorescence spectra were obtained with an Hitachi F-4500 spectroflurometer. Excitation and emission bandwidths of 2.5 and 5.0 nm were employed, respectively, and a scan speed of 240 nm/min was used. Protein concentrations of 1.15 and 2.3 M were used in 1 times 0.1-cm cuvettes. Samples were excited at 295 nm to produce > 95% emission from Trp residues. All spectra were corrected for photomultiplier response and Raman scattering.

Equilibrium Sedimentation

Equilibrium sedimentation was performed with a Beckman Optima XLA analytical ultracentrifuge. Experiments were conducted at 16,000 and 22,000 rpm in an An60Ti rotor at a protein concentration of 0.6 M at 4 °C, using complexes formed under conditions identical to those used in the circular dichroism and fluorescence experiments. Data were analyzed by a nonlinear fitting program provided by the National Ultracentrifugation Center at the University of Connecticut.

IL-5-dependent Cell Proliferation Assay

The murine B-cell lymphoma BCL-1 (obtained from ATCC) was passaged five times in RPMI 1640 cell culture medium supplemented with 20% fetal bovine serum in the presence of penicillin and streptomycin. The cells were harvested by gentle scraping, washed once, and resuspended in serum-free medium (Kemp Biotechnologies; Cyto SF-4) containing 0.1% bovine serum albumin at a concentration of 0.5 times 10^6 cells/ml. Aliquots (60 µl) were transferred to a flat-bottomed microtiter plate and preincubated at 37 °C. Recombinant human IL-5 (0-500 nM) was mixed with purified alphaRED (0, 30, 100, or 300 nM) in quadruplicate aliquots in serum-free medium in a polypropylene microtiter plate. Receptor and ligand were incubated for 30 min at 37 °C and 95% humidity, and then 60-µl aliquots mixed with the preincubated BCL-1 cells. Cellular proliferation of BCL-1 in the presence/absence of rhIL-5/alphaRED was assayed after 17 h at 37 °C and 95% humidity by the conversion of a tetrazolium salt MTT (Promega) to formazan over 8 h, and quantitated by measurement of optical density at 570 nm. Prior experiments have shown that this technique was essentially equivalent to [^3H]thymidine incorporation in these cells (data not shown).


RESULTS

Engineering of Epitope-tagged Soluble IL-5 Receptor alpha Chains

Fig. 1illustrates the cloning strategy employed to engineer the three soluble isoforms of the ligand-binding extracellular domain bearing C-terminal epitope tags of eight amino acids (FLAG, IBI Inc.) from the cDNA corresponding to the full-length IL-5 receptor alpha chain cDNA. The S1-alphaRED and S2-alphaRED constructs correspond to the S1 and S2 nomenclature previously assigned to the two alternatively spliced mRNA species identified encoding for soluble variants of the full-length transmembrane IL-5 receptor alpha chain(19) . The S3-alphaRED construct corresponds to the entire extracellular ligand-binding domain of the IL-5 receptor up to but not including the first amino acid in the putative transmembrane domain (Fig. 1A; (3) ). A single oligonucleotide, DN265, when used to prime the PCR reactions incorporated a BamHI restriction site into the cDNA as well as a consensus sequence for introducing a favorable context around the AUG initiator codon for optimal translation (Fig. 1B; (20) ). Three different oligonucleotides (PB20, PB23, and PB26) were paired with DN265 to introduce the appropriate coding sequences at the 3` end of the cDNA to encode the C-terminal amino acids of the soluble S1-, S2-, and S3-alphaRED. A final round of PCR reactions paired oligonucleotide DN265 to the three oligonucleotides PB22, PB25, and PB27 to introduce the sequence encoding the eight amino acid FLAG epitope tag, two stop codons and a BamHI restriction site (Fig. 1C). The final three cDNAs were then cloned into BamHI-digested baculovirus transfer vector pETL as BamHI fragments behind the polyhedrin promoter and then recombinant virus was generated by in vivo homologous recombination in Sf9 cells as described under ``Experimental Procedures.'' There are 10 noncoding bases present at the 5` end of the alphaRED cDNA when introduced as a BamHI fragment before the initiator methionine of the alphaRED.


Figure 1: Engineering of epitope-labeled alphaRED. Panel A, a schematic representation of the full-length human IL-5 receptor alpha chain (IL-5Ralpha) polypeptide shows the ligand-binding domain (shaded box), the transmembrane domain (hatched box), and the intracellular domain (open box). The SacI restriction site () was used to reconstruct the full-length cDNA from two PCR-derived clones (see ``Experimental Procedures''). The position of the IL-5 receptor chain amino acid at the junction with the sequence encoding the FLAG epitope is indicated for the S1, S2, and S3 forms of the soluble receptor. The numbers refer to the amino acid residue with reference to the N terminus. Panel B, the nucleotide sequence and deduced amino acid sequence of the 5`-region of the three soluble receptor isoforms is shown with that part corresponding to the PCR-priming oligonucleotide DN265 which is boxed. Panel C, the nucleotide sequences and deduced amino acid sequences of the 3`-regions of the three soluble receptor isoforms are shown with those parts corresponding to the PCR-priming oligonucleotides PB22, PB25, and PB27 (boxed) used to generate the S1-, S2-, and S3-alphaRED constructs, respectively.



Baculovirus Expression of the alphaRED-FLAG

Insect cells in log phase of growth (1-1.5 times 10^6 cells/ml) were infected with high titer recombinant virus stock at an multiplicity of infection of approximately 3 under low serum conditions (0.5% FBS). When infected at a cell density of 2-3 times 10^6 cells/ml, 3-5 mg/liter alphaRED was routinely obtained in a single-step affinity purification. Fig. 2illustrates the time course of expression of the three isoforms of the soluble IL-5 receptor. Immunoreactive material was evident at 48 h postinfection both in the Triton-solubilized cell lysate as well as in the media. The media were routinely harvested at 72 h postinfection when greater than 90% of the cells excluded trypan blue, but significant cell death (and the production of associated proteolytic enzymes) had not yet occurred. The S1-alphaRED and S2-alphaRED proteins appear to be efficiently processed and secreted as evidenced by the lack of a large amount of cell-associated immunoreactive material at the various time points. The S3-alphaRED, on the other hand, consistently expressed to approximately 50% of the yield of the S1- and S2-alphaRED constructs and displayed approximately 50% of the total immunoreactive material associated with the cells (Fig. 2C). The S3-alphaRED protein did not appear to be associated with the cell membrane of the infected insect cells, nor was a large amount of the intracellular material capable of binding IL-5 since affinity cross-linking of S1-, S2-, and S3-alphaRED recombinant virus-infected cells displayed equivalent low levels of I-IL-5 binding to intact infected insect cells. Triton X-100 (1%) lysates of pETL-S1-, S2-, and S3-alphaRED construct-infected insect cells bound I-IL-5 to equivalent levels indicating that the large amount of cell-associated immunoreactive material in the S3-alphaRED cell lysates is incapable of binding IL-5 (data not shown).


Figure 2: Time course of baculovirus expression of alphaRED isoforms. Conditioned media (M) and Sf9 lysates (C) obtained 0, 24, 48, and 72 h after infection were subjected to immunoblot analysis using the FLAG M2 monoclonal antibody (see ``Experimental Procedures'').



IL-5 Binding of the alphaRED

The affinity of the alphaRED (S1-,S2-, and S3-) for IL-5 was estimated by affinity cross-link labeling of the alphaRED in the conditioned media prior to purification. Fig. 3demonstrates that 2-5 nM rhIL-5 is required to compete 50% of 0.5 nMI-IL-5 from a receptor solution of approximately 0.5 nM. The results of densitometric scanning of the autoradiograms in Fig. 3A are shown in Fig. 3B and illustrates that the affinity of the three isoforms of the alphaRED appear identical.


Figure 3: Affinity cross-link labeling of I-rhIL-5 to S1-S2- and S3-alphaRED. Panel A, aliquots of medium harvested 72 h postinfection with each of the baculovirus constructs were incubated with 0.5 nMI-rhIL-5, and unlabeled rhIL-5 (0, or 30 pM to 200 nM) as described under ``Experimental Procedures.'' An aliquot (50-fold dilution) of each was subjected to SDS-PAGE and autoradiography to assess competition. Panel B, the resulting autoradiograms were scanned with a laser densitometer (Molecular Devices) and the integral of the density of the bands corresponding to the ligand-receptor complex obtained. The values were expressed as a percentage of the labeling in the absence of competing IL-5, and the EC for competition calculated by linear interpolation.



Affinity Purification of the alphaRED

Fig. 4A is a representative immunoblot analysis of a purification of approximately 350 mg of S1-alphaRED material from 100 ml of 2.6 times 10^6 cells/ml harvested at 72 h postinfection. A single pass of the titrated, filtered conditioned medium over the affinity matrix depleted the media of the alphaRED (S1-alphaRED; Fig. 4A, lane 1 (before purification) and lane 2 (after a single pass over the affinity column)). The column was washed extensively (see ``Experimental Procedures'') and the S1-alphaRED eluted with 100 mM glycine to >95% purity (Fig. 4A, lanes 3-10) on silver-stained SDS-PAGE gels (data not shown). The purified material was heterogeneous and ran as a doublet under reducing SDS-PAGE. The heterogeneity of the alphaRED was due to differential glycosylation since enzymatic deglycosylation with N-glycosidase F reduced the doublet to a single band of approximately 40.3 kDa apparent molecular mass (Fig. 4B, lane 1 (before deglycosylation) and lane 2 (after deglycosylation)).


Figure 4: Purification of S1-alphaRED FLAG from 72 h postinfection conditioned medium. Panel A, conditioned medium from Sf9 cells infected with the S1-FLAG construct prior to purification (lane 1), after a single pass over the affinity column (lane 2) and fractions eluted from M2 anti-FLAG immunoaffinity column with 100 mM glycine, pH 3.0 (lanes 3-10) were subjected to SDS-PAGE immunoblot analysis. Panel B, deglycosylation of purified S1-alphaRED FLAG with 2 units of N-glycosidase F for 16 h at 22 °C in the absence (lane 1) and presence (lane 2) of the enzyme.



Biophysical Measurements

To ensure that a complex was actually forming under the conditions of the circular dichroism (CD) and fluorescence experiments, the molecular weight of a 1:1 molar ratio mixture of IL-5 and the extracellular receptor domain (S1-alphaRED) was analyzed by equilibrium sedimentation analysis at a protein concentration of 0.6 M. The soluble receptor alone had an estimated molecular mass of 39.5 kDa under these conditions. Molecular masses of 70 and 74 kDa were found at two different rotor speeds, consistent with the expected value of approximately 70 kDa for the formation of a ligand-receptor complex.

To evaluate the secondary structure of the alphaRED, its far-UV circular dichroism spectrum was obtained between 195 and 260 nm (Fig. 5). The spectrum manifested a broad, weak negative double minimum at 210 and 200 nm. Analysis of this spectrum by the self-consistent singular value decomposition algorithm of Sreerama and Woody (18) yielded an estimated secondary structure content of 42% beta-sheet, 20% turn, 32% disordered, and 6% alpha-helix. Thus, this portion of the IL-5 receptor appears to consist primarily of beta-structure. Also shown in Fig. 5is the CD spectrum of IL-5, which displayed the characteristic double minimum at 208 and 222 nm expected for this alpha-helix rich protein. When a 1:1 molar complex was formed between IL-5 and the extracellular portion of its receptor, the resulting spectrum was dominated by the much stronger spectrum of the interleukin (Fig. 5). When the spectrum of the complex was compared to the numerical sum of the spectra of the individual spectra of IL-5 and the receptor domain, little difference was seen. This suggests that no major reorganization of secondary structure occurs in either protein when the complex is formed.


Figure 5: Circular dichroism analysis of IL-5/alphaRED interactions. The far-UV circular dichroism spectrum of recombinant human IL-5 gave the characteristic profile of this species (bulletbulletbullet). The recombinant IL-5 receptor extracellular domain construct S1-alphaRED spectrum(- - -) suggested that the major structural feature of this protein was beta-sheet (42%), with only 6% consisting of alpha-helix (see ``Results''). The spectra of a 1:1 molar complex of the receptor domain and IL-5 (-) and the theoretical sum of the individual spectra of the receptor domain and IL-5(- - -) were essentially superimposable, implying that the conformational changes occurring on receptor-ligand interaction were modest and subtle. The spectra of the free receptor domain and IL-5 were obtained at a protein concentration of 2.3 M, while that of the complex and the mathematical sum at 1.15 M. The spectrum of the receptor domain is multiplied by 4 for illustration purposes. All spectra were measured in 6 mM sodium phosphate, 0.15 M NaCl, pH 7.0, at 22 °C.



To probe the possibility that a change in stability might occur when the two proteins bind, the thermally induced unfolding of IL-5, the receptor domain and the complex was measured by monitoring the CD at 208, 215, 222, 230, and 350 nm as a function of temperature. The CD spectrum of the complex was dominated by that of IL-5, and thus only a change in stability of this component could be unambiguously detected by this method. In fact, both the complex and IL-5 itself displayed midpoints of their unfolding temperatures at 72-74 °C (not illustrated), implying that there was no major change in the thermal stability of the IL-5 component upon complex formation.

To determine if an alteration in tertiary structure might be induced by complex formation, the tryptophan fluorescence emission spectra of the same proteins were examined. The IL-5 receptor ectodomain (S1-alphaRED) displayed a fluorescence emission maximum at approximately 338 nm, suggesting that its nine tryptophan residues are on the average relatively buried (Fig. 6). The single tryptophan of rhIL-5 appeared somewhat more exposed, manifesting a fluorescence peak at 345 nm. The complex produced a maximum at an intermediate location near 342 nm. No difference was seen when the fluorescence emission peak of the complex was compared to the numerical sum of the peaks of the receptor domain and IL-5 themselves, again suggesting little structural alteration upon complex formation (Fig. 6).


Figure 6: Spectrofluorimetric analysis of IL-5/alphaRED interactions. Intrinsic fluorescence emission spectra of IL-5 (bulletbulletbullet), the recombinant IL-5 receptor extracellular domain, (- - -), a 1:1 molar complex of the receptor domain and IL-5 (-), and the theoretical sum of the individual spectra of the receptor domain and IL-5(- - -) are shown. Protein concentrations and solution conditions were the same as those shown in Fig. 5. The intensity of the spectra of the complex and the sum of the proteins were divided by 2 to enhance comparison. Proteins were excited at 295 nm to produce primarily tryptophan emission and are corrected for Raman scattering.



Nonequilibrium Binding of alphaRED to IL-5

The kinetics of binding of the purified alphaRED to IL-5 were studied under conditions of nonequilibrium binding with an analytical biosensor with detection based on the optical phenomenon of surface plasmon resonance(21) . The BIAcore(TM) system has been used previously to study various types of molecular interactions including those involving antigen/antibodies, signal transducing complex formation, and phosphorylated peptide/SH2 domains. Direct coupling of IL-5 to the sensor chip was found to be the most efficient use of reagents since the IL-5 surface could be regenerated with 20 mM HCl more than 30 times, while retaining receptor binding capabilities. Each IL-5 surface was regenerated only 20 times before preparing a fresh surface.

Fig. 7(panel A) shows an overlay of the sensorgrams obtained after interaction of various concentrations of the purified S1-alphaRED protein with the IL-5 sensor-chip surface as described under ``Experimental Procedures.'' Panel B illustrates the specificity of the alphaRED interaction with IL-5 since preincubation of the receptor solution with IL-5 reduced the interaction of the soluble receptor with the IL-5 sensor. With this experimental format, the K(d) of the alphaRED was calculated using the association and dissociation rate constants (2.8 times 10^6M s and 2.56 times 10 s, respectively) derived from the BIAcore sensorgrams and the equation K(d) = k/k to be 9 nM at 25 °C. This value did not differ significantly from values determined for the S2- and S3-alphaRED forms (data not shown). The effects of various conditions for the elution of S1-alphaRED from the affinity column on the binding interactions of the resulting purified alphaRED proteins were also studied using the IL-5 surface. No differences in the association or dissociation rate constants were detected when the S1-alphaRED was eluted either with 100 mM glycine, pH 3.0, or with an excess of the FLAG peptide (data not shown), suggesting that exposure to low pH did not significantly affect the tertiary structure of these proteins. Accordingly, glycine elution was utilized for all additional experiments, as no further separation (from the FLAG peptide) was required.


Figure 7: BIAcore analysis of non-equilibrium analysis of IL-5/alphaRED interactions. Panel A, the relative mass response (vertical axis, arbitrary units) was measured for the interaction of purified S1-alphaRED FLAG (90 nM to 1.35 µM) with immobilized rhIL-5 (see ``Experimental Procedures''). At 100 s (horizontal axis), PBS control buffer (5 ml/min) was replaced with buffer containing the recombinant receptor to assess the association rate of the complex. After 340 s, this was in turn replaced by the control buffer to determine the dissociation rate. The IL-5 surface was regenerated after each exposure to the receptor (see ``Experimental Procedures''). Repeated regeneration had no effect on the kinetics or magnitude of interactions. Panel B, the relative mass response (vertical axis, arbitrary units) was measured as above for the interaction of 230 nM of the alphaRED in the absence (upper trace) and presence (lower trace) of an excess (500 nM) of IL-5. In the latter experiment, the high affinity association was abolished, demonstrating the specificity of the response. Panel C, the slopes (vertical axis) of the association phase curves for each alpha chain concentration (panel A) were plotted against the mass response (R; horizontal axis) for 10 s after exposure to the soluble receptor at 100 s. The slope of each individual plot represents the negative value for k(s) at each concentration of the S1-alphaRED FLAG construct. Panel D, the values for k(s) were then plotted (vertical axis) against the receptor concentration (horizontal axis; nM), and an association rate constant (k or k) of 2.8 times 10^6M s derived from the slope of the line of best fit. An equivalent analysis (see ``Experimental Procedures'') for the dissociation phase of the analysis (after 350 s; panel A) resulted in a dissociation rate constant (k or k) of 2.56 times 10 s. An equilibrium dissociation constant (K) of 9 nM was then calculated from k/k)



IL-5-dependent Cell Proliferation Assay

Murine BCL-1 lymphoma cells demonstrated a dose-dependent proliferative response to rhIL-5 (Fig. 8) with an EC of 16.7 nM. Preincubation of the rhIL-5 with 30, 100, or 300 nM alphaRED resulted in a dose-dependent rightward shift of the IL-5 dose-response curve, resulting in EC values of 16.8, 55.2, and 62.5 nM, respectively. At fixed concentrations of 5 and 20 nM rhIL-5, the soluble receptor dose-dependently inhibited the resultant proliferative response, achieving 100 and 49% inhibition, respectively, at 300 nM.


Figure 8: Inhibition of rhIL-5-induced BCL-1 proliferation by alphaRED. 3 times 10^4 murine BCL-1 cells (bullet) responded by proliferation (MTT conversion measured by OD at 550 nm; vertical axis) to the presence of rhIL-5 (0-500 nM; horizontal axis). Preincubation (see ``Experimental Procedures'') with the purified S1-alphaRED at 30 nM (circle), 100 nM (box), and 300 nM (up triangle) shifted the IL-5 response curve to the right. Values are the mean ± S.E. of 4-6 replicates from one of three separate experiments with three S1-alphaRED preparations. Inset, The IL-5 proliferative response (vertical axis) at 5 nM (box) and 20 nM () agonist was plotted against the concentration of neutralizing S1-alphaRED to demonstrate dose dependence.




DISCUSSION

Eosinophilic pulmonary inflammation has been recognized as a hallmark of chronic bronchial asthma for over 100 years, and the presence of eosinophils in the lung has recently been correlated with the degenerative pathophysiology characteristic of this disease(22) . The identification of IL-5 as a potent in vitro eosinophilopoietin(1) , is consistent with the ability of IL-5 overexpression in transgenic mice to induce tissue eosinophilia (23, 24) and of IL-5 antibodies to inhibit pulmonary eosinophilia in animals (25) and suggests that this cytokine plays a critical role in the development and tissue targeting of pro-inflammatory eosinophils. Thus, disruption of the actions of interleukin-5 offers a significant opportunity for novel therapeutic intervention in asthma. Accordingly, we have used the baculovirus expression system to produce recombinant epitope-labeled form of the extracellular domain of the human IL-5 receptor to allow detailed analyses of the binding and biological interactions of this component of IL-5 function.

The cDNA encoding the full-length IL-5 receptor alpha chain (Fig. 1A) was isolated from a strain of eosinophilic human erythroleukemic cells, shown previously to possess high affinity IL-5 receptors, and to be activated by pM concentrations of rhIL-5(26) . Alternative isoforms corresponding to soluble forms of the extracellular domain of the alpha chain truncated prior to the transmembrane spanning region (3) were then used to engineer three epitope-labeled forms of the ligand-binding domain of the alpha chain (Fig. 1, B and C). Transfection of these constructs into Sf9 cells using recombinant baculoviral vectors resulted, after 48 h, in the secretion into the medium of 43-kDa molecular mass proteins bearing the FLAG epitope (demonstrated by immunoblot; Fig. 2). In the case of the S1- and S2-alphaRED constructs, the protein was predominantly secreted, with minimal immunoreactive tagged protein being detectable in insect cell lysates. This high level of expression and secretion of the alphaRED allowed demonstration of IL-5 binding directly in conditioned medium by affinity cross-link labeling. In these experiments, the ED for competition of 0.5 nM of I-IL-5 binding was found to be 2-5 nM, with no significant differences observed between the various forms (Fig. 3).

The relatively high level of expression and secretion of the alphaRED also allowed rapid and mild immunoaffinity purification with an anti-FLAG column (Fig. 4A). Glycine-eluted material was heterogeneously glycosylated, the extent of which did not appear to affect ligand binding. The affinity-purified S1-alphaRED was found by sedimentation analysis to form a 1:1 ligand-receptor complex with a 70-74-kDa molecular mass as predicted, demonstrating the utility of epitope-tagging in the production of a soluble cytokine receptor subunit.

Circular dichroism analysis (Fig. 5) and intrinsic fluorescence emission spectroscopy (Fig. 6) indicated that the S1-alphaRED existed with a defined secondary and tertiary structure, which could be altered to a substantially disordered form by elevated temperatures. The S1-alphaRED consisted primarily of beta-structure with a substantial portion of its Trp residues buried in the protein's interior. Comparison of the spectra of the interleukin/receptor domain complex to that of the mathematical sum of the free ligand and receptor suggested that little structural alteration occurred upon complex formation (although small alterations in side chains might not be detectable by these analyses). This absence of major conformational change upon interaction with the ligand implies that direct signal transduction is unlikely to occur via a membrane-associated alpha chain alone, but requires further association with the beta-subunit to activate the latter's large intracellular domain. The alphaRED proteins were also amenable to biophysical analysis using surface plasmon resonance detection. Analysis of the kinetic parameters derived from non-equilibrium binding using this technique (Fig. 6) gave a calculated dissociation constant of 9 nM at 25 °C. This value is within 2-fold of that estimated from the affinity cross-link labeling experiments (which were performed at 4 °C), and is consistent with an increase in affinity with reduced temperature as shown by microcalorimetric determinations(21) . Indeed, this value is in excellent agreement with that reported in analogous immobilized IL-5 experiments (5.5 nM) for the non-tagged soluble alpha chain expressed in Drosophila cells(21) . Thus, the process of FLAG epitope tagging does not appear to affect IL-5/alphaRED interactions, although adding tremendous utility for the expression and purification of these, and additionally mutated forms of the soluble domain.

The affinity-purified alphaRED described above showed significant inhibitory activity against an IL-5-induced proliferative response in the murine B-cell lymphoma BCL-1 in a serum-free medium (Fig. 8). At a 60-fold molar excess, the soluble form of the receptor completely ablated the proliferative response to IL-5 (Fig. 8, inset). This is consistent with the suggestion that naturally occurring soluble forms of cytokine receptors act to modulate the biological activity of these potent hematopoietins. Whereas the potency of this modulation in the BCL-1 bioassay was significantly less than that observed for the avidity of the soluble receptor for IL-5 as measured by affinity cross-link labeling, and non-equilibrium interactions, little is known about the stability of this recombinant protein in cellular incubates, or the degree or duration of IL-5 receptor occupancy required for signal transduction. In this respect, a large molar excess of the soluble receptor may be necessary to completely inhibit the biological response to IL-5 by vastly reducing the frequency of reversible IL-5-receptor interactions which could otherwise trigger irreversible cellular proliferation.

In summary, we have engineered three epitope-tagged soluble forms of the extracellular domains of the IL-5 receptor alpha-subunit and demonstrated their equivalent abilities to interact with rhIL-5 at nanomolar concentrations. After simple affinity purification, one of these isoforms was shown to inhibit the proliferative activity of rhIL-5 on murine B-lymphoma cells, suggesting that this novel construct itself may have therapeutic potential in the treatment of eosinophilic inflammation. Furthermore, circular dichroism and fluorescence spectral analysis of this recombinant protein demonstrated that the conformational changes associated with ligand binding were comparatively minor and subtle. Therefore, with additional structural information, it appears possible that molecules could be designed to disrupt the interaction of IL-5 with its receptor and thus act as IL-5 receptor antagonists.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 514-428-8544; Fax: 514-695-0693.

(^1)
The abbreviations used are: IL, interleukin; IL-5alphaR, IL-5 receptor alpha-subunit; IL-5betaR, IL-5 receptor beta-subunit; alphaRED, extracellular domain of the IL-5 receptor alpha-subunit; PCR, polymerase chain reaction; FLAG(TM), N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C octapeptide; BS^3, bis(sulfosuccinimidyl) suberate; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Hugh Song for assistance with large scale purification of the S1-FLAG construct, Kevin Neden for the provision of BCL-1 cells, Dr. Brian Kennedy for HL-60 total RNA, and Dr. Jilly Evans for critical reading of the manuscript.


REFERENCES

  1. Sanderson, C. J. (1992) Blood 79, 3101-3109 [Medline] [Order article via Infotrieve]
  2. Tavernier, J., Devos, R., Cornelis, S., Tuypens, T., Van der Heyden, J., Fiers, W., and Plaetinck, G. (1991) Cell 66, 1175-1184 [Medline] [Order article via Infotrieve]
  3. Murata, Y., Takaki, S., Migita, M., Kikuchi, Y., Tominaga, A., and Takatsu, K. (1992) J. Exp. Med. 175, 341-351 [Abstract]
  4. Fishkoff, S. A. (1988) Leuk. Res. 12, 679-686 [CrossRef][Medline] [Order article via Infotrieve]
  5. Scheid, M., Metters, K. M., Slipetz, D. M., Cuncic, C. F., Ali, A., Brown, P., Van Riper, G., Rosen, H., and Nicholson, D. W. (1994) Can. J. Physiol. Pharmacol. 72, Abstr. 281
  6. Brown, P. M., Scheid, M. P., O'Neill, G. P., Tagari, P., and Nicholson, D. W. (1995) Protein Exp. Purif. 6, 1-9 [CrossRef][Medline] [Order article via Infotrieve]
  7. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  8. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. (1990) PCR Protocols: A Guide to Methods and Applications , Academic Press, New York
  9. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Nucleic Acids Res. 19, 1154 [Medline] [Order article via Infotrieve]
  10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, Cold Spring Harbor, NY
  11. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual , W. H. Freeman and Co., New York
  12. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  13. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  14. Thotakura, N. R., and Bahl, O. P. (1987) Methods Enzymol. 138, 350-359 [Medline] [Order article via Infotrieve]
  15. Jonsson, U., Fagerstam, L., Roos, H., Ronnberg, S., Sjolander, S., Stenberg, E., Stahberg, R., Urbaniczky, C., Ostlin, H., and Malmqvist, M. (1991) BioTechniques 18, 620-627
  16. Karlsson, R., Michaelsson, A., and Mattsson, L. (1991) J. Immunol. Methods 145, 229-240 [CrossRef][Medline] [Order article via Infotrieve]
  17. Panayotou, G., Gish, G., End, P., Truong, O., Gout, I., Dhand, R., Fry, M. J., Hiles, I., Pawson, T., and Waterfield, M. D. (1993) Mol. Cell. Biol. 13, 3567-3576 [Abstract]
  18. Sreerama, N., and Woody, R. W. (1993) Anal. Biochem. 209,, 32-44 [CrossRef][Medline] [Order article via Infotrieve]
  19. Tavernier, J., Tuypens, T., Plaetinck, G., Verhee, A., Fiers, W., and Devos, R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7041-7045 [Abstract]
  20. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148 [Abstract]
  21. Morton, T. A., Bennett, D. B., Appelbaum, E. R., Cusimano, D. M., Johanson, K. O., Matico, R. E., Young, P. R., Doyle, M., and Chaiken, I. M. (1994) J. Mol. Recog. 7, 47-55
  22. Ohashi, Y., Motojima, S., Fukuda, T., and Makino, S. (1992) Am. Rev. Respir. Dis. 145, 1469-1476 [Medline] [Order article via Infotrieve]
  23. Dent, L. A., Strath, M., Mellor, A. L., and Sanderson, C. J. (1990) J. Exp. Med. 172, 1425-1431 [Abstract]
  24. Kruger-Krasagakes, S., Li, W., Richter, G., Diamantstein, T., and Blankenstein, T. (1993) Eur. J. Immunol. 23, 992-995 [Medline] [Order article via Infotrieve]
  25. Gulbenkian, A. R., Egan, R. W., Fernandez, X., Jones, H., Kreutner, W., Kung, T., Payvandi, F., Sullivan, L., Zurcher, J. A., and Watnick, A. S. (1992) Am. Rev. Respir. Dis. 146, 263-265 [Medline] [Order article via Infotrieve]
  26. Tagari, P., Pecheur, E. I., Scheid, M., Brown, P., Ford-Hutchinson, A. W., and Nicholson, D. W. (1993) Int. Arch. Allergy Clin. Immunol. 101, 227-233

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