From www.bloodjournal.org by guest on January 21, 2015. For personal use only. Monoclonal Antibody 7 6 3 Recognizes the N-Terminal Domain of the Human Interleukin-3 (IL-3) Receptor a-Chain and Functions as a Specific IL-3 Receptor Antagonist By Qiyu Sun, Joanna M. Woodcock, Aaron Rapoport, Frank C. Stomski, Eija I. Korpelainen, Christopher J. Bagley, Gregory J. Goodall, William B. Smith, Jennifer R. Gamble, Mathew A. Vadas, and Angel F. Lopez The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading t o cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute t o pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds t o the IL-3R a-chain and completely abolishes its function. MoAb 763 immunoprecipitated and recognized in Western blots the IL-3R achain expressed by transfected cells and bound t o primary cells expressing IL-3Ra. MoAb 7G3 bound the IL-3R a-chain with a kd of 900 pmol/L and inhibited '**I-IL-3 binding t o high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited lZ51-7G3binding t o high- and low-affinity IL-3Rs. indicating that MoAb 7G3 and IL-3 bind t o common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 763 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell prolideration, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R a-chain MoAbs failed t o inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R a-chain mutants and IL-3RaIGM-CSFRa chimeras revealed that 31 amino acids in the N-terminus of IL-3Ra were required for MoAb 763 binding. MoAb 763 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the Nterminal domain of IL-3Ra in IL-3 binding. Since this domain is unique t o the IL3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors. 0 1996 by The American Society of Hematology. H domains." In addition, there is also an N-terminal domain, which, interestingly, has sequence similarities with human granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5 receptor a - c h a k 2 ' This feature distinguishes these receptors from the other members of the cytokine receptor family. The functions of the CRM and Nterminal domain of the I L 3 R a-chain are not known, nor is it known where the IL-3 binding regions lie in the receptor. We show here the characterization of a monoclonal antibody (MoAb), 7G3, directed against the IL-3R a-chain, which is capable of inhibiting IL-3 binding and antagonizing IL-3 functions. The MoAb 7G3 epitope maps to amino acids 19 to 49 of the N-terminal domain of IL-3R a-chain. These results offer the potential to block IL-3 activity in vivo and suggest that the N-terminal domain of IL-3Ra, and by anal- UMAN interleukin-3 (IL-3) is a pleiotropic cytokine that stimulates production of hematopoietic cells from multiple lineages, including neutrophils, eosinophils, monocytes, megakaryocytes, erythroid cells, basophils, and B cells.'-6 Recently, IL-3 has also been shown to regulate vascular endothelial cell functions, enhancing adhesion molecule expression, neutrophil transmigration, and cytokine prod~ction.~,' Although some of the effects of IL-3 may be desirable and have prompted its clinical use in bone marrow reconstitution following chemotherapy,' it is also apparent that abnormal or excessive production of IL-3 has the potential to lead to disease states. For example, some acute myeloid leukemias proliferate in response to IL-3,'0.i' and cells from follicular B-cell lymphomas produce and depend on IL-3 for their growth.I2 IL-3 has also been implicated in allergy, not only for its ability to stimulate eosinophil and basophil p r o d ~ c t i o n but ~ ~ 'also ~ for being a strong stimulus of histamine release from basophils in vitr0.4~'~ Detection of elevated amounts of IL-3 mRNA in the skin and bronchi of allergic individualsi5 further suggests an in vivo role in allergy. The biologic activities of human IL-3 are initiated by the binding of IL-3 to its receptor (IL-3R). This consists of two subunits: an a-chain (IL-3Ra) that binds IL-3 specifically and with low affinity,16 and a P-chain (Pc)that does not bind ligand on its own but confers high-affinity binding when co-expressed with I L - ~ R C ~ . 'Both ~ , ' ' chains are required for signalingI8;however, receptor activation and cellular signaling are dependent on IL-3 binding to IL-3Ra as the initial step. The subsequent events are not fully understood, but probably involve receptor dimerization leading to the activation of specific kinases associated with the receptor." The structure of the extracellular domain of human IL3Ra has not yet been elucidated. Since IL-3Ra belongs to the cytokine receptor family, it is predicted to contain a cytokine receptor module (CRM) with two discrete folding Blood, Vol 87, No 1 (January 1). 1996: pp 83-92 From the Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia: and the Hematology Unit, University of Rochester Medical Center, Rochester, NY. Submitted July 3, 1995; accepted August 15, 1995. Supported by grants from the National Health and Medical Research Council ofAustralia. Q.S.is a recipient of a Dawes Postgraduate Scholarship from the Royal Adelaide Hospital. C.J.B. is a Rotary Peter Nelson fellow of the Anti-Cancer Foundation of the University of South Australia. A.R. was supported by fellowships from the Leukemia Society of America and the James R. Wilmot Foundation. Address reprint requests to Angel F. Lopez, MD, PhD, Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5OOO. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8701-0016$3.00/0 83 From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 84 SUN ET AL ogy those also of GM-CSFR and IL-SR a-chains, may be involved in ligand binding. MATERIALS AND METHODS Cell lines, media, and cytokines. The CHO cell line, F6, expressing the IL-3R a-chain was developed for screening and characterization of anti-IL-3Ra MoAbs. Briefly, IL-3R a-chain cDNA was cloned into pcDNA1Neo (Invitrogen, San Diego, CA) and transfected into CHO cells by electroporation.22COS cells transiently transfected with IL-3R a-chain cDNA by electr~poration’~ with or without p-chain cDNA were used for immunization and characterization of anti-IL-3R a-chain MoAb. TF-1 cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and GM-CSF (2 ng/mL). GM-CSF was a gift from Genetics Institute (Cambridge, MA). Recombinant human IL-3 was produced in Escherichia coli as previously de~cribed.’~ Generation of MoAbs. BALBlc mice were immunized intraperitoneally with 5 X lo6 COS cells transfected with the IL-3R a-chain in combination with 100 pg Adjuvant Peptides (Sigma, St Louis, MO). This procedure was repeated three times at 2-week intervals. Four weeks after the final immunization, a mouse was boosted intravenously with 2 X 106COS cell transfectants. Three days later, the splenocytes were fused with the mouse NS-1 myeloma cells at the ratio of 4: 1 using 50% polyethylene glycol as previously described.” After fusion, the cell suspension was cultured in RPMI 1640 supplemented with 20% FCS and 20% 5774 conditioned medium.26Hybridoma cells were selected with hypoxanthine-aminopterin-thymidine. Hybridoma supernatants were screened using F6 cells by an antigencapture immunoassay with Rose Bengal as a colorimetric indicator.” Positive clones were subcloned by limiting dilution, and the culture conditions were gradually reduced to RPMI 1640 complete media supplemented with 10% FCS. Antibody-containing ascites fluid was produced by injecting the hybridoma cells into pristane-treated mice. MoAbs were purified from ascites fluid on a protein A-Sepharose column (Pierce, Rockford, IL) as described by the manufacturer. MoAbs were isotyped by means of a mouse-hybridoma subtyping kit (Boehringer Mannheim, Germany). Immunofluorescence. Freshly purified neutrophils, eosinophils, monocytes, human umbilical cord venular endothelial cells (HUVEC), or F6 cells (5 X lo5) were incubated with 50 pL hybridoma supernatant or 0.25 pg purified MoAb for 45 to 60 minutes at 4°C. Cells were washed twice and then incubated with FTTC-conjugated rabbit anti-mouse Ig (Silenus, Hawthorn, Victoria, Australia) for another 30 to 45 minutes. Fluorescence intensity was analyzed on an EPICS-Profile I1 Flow Cytometer (Coulter Electronics, Hialeah, FL). In experiments with truncated IL-3R a-chain and IL-3RalGMCSFRa chimera, transfected COS cells were examined under a fluorescence microscope. Immunoprecipitarions. F6 cells (4 X 10’) were surface-labeled using Na ‘’’1 (New England Nuclear, Boston, MA) as previously described.**Cells were washed three times with phosphate-buffered saline and lysed in 1 mL RIPA buffer with protease inhibitors (25 mmol/L Tris hydrochloride, pH 7.4, 150 mmoVL NaC1, 1% Triton, 0.5% deoxycholate, 0.05% sodium dodecyl sulfate (SDS), 2 mmol/ L PMSF, 10 mmom soybean trypsin inhibitor, 20 mmol/L leupeptin, and 5% aprotonin [Sigma]). The cell extracts were centrifuged at 10,OOO x g for 15 minutes, and the cell lysates were precleared twice with protein A-Sepharose before incubating 250 pL lysates with MoAbs (2 pg/mL) overnight at 4°C. Protein A-Sepharose was then added, and bound proteins were washed with RIPA buffer, eluted with SDS loading buffer with 2-mercaptoethanol, and analyzed by 10% SDS-polyacrylamide gel electrophoresis (PAGE). Radiolabeled proteins were visualized using an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Western blotting. F6 cells (3 X 10’) were solubilized in reducing SDS loading buffer, and proteins were separated by 10% SDS-PAGE before transfemng electrophoretically onto nitrocellulose filters. Filters were then blocked with TNT buffer (10 mmoVL Tris hydrochloride, pH 8.0, 150 mmol/L NaCI, and 0.05% Tween 20) containing 3% gelatin. Anti-IL-3Ra MoAbs (2 pg/mL) were diluted in TNT buffer containing 1% gelatin and incubated with the filters for 90 minutes. The filters were then incubated with ‘251-proteinA (New England Nuclear) for 45 minutes and washed thoroughly with TNT buffer. Radiolabeled proteins were detected as described earlier. For Western blot analysis of truncated and chimeric IL-3R a-chains, gels were electroeluted onto PVDF membrane and filters blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (‘ITBS). Filters were incubated with MoAbs (1 to 10 pg/ mL) in TTBS with 5% BSA for 2 hours. Flag-tagged proteins were detected with 3 pg/mL anti-flag MoAb M2 (IBI, New Haven, CT). The secondary antibody, alkaline phosphatase-tagged goat antimouse (Pierce) was then added at a dilution of 1:2,500 in ‘ITBS. MoAb-bound proteins were visualized using the BCIPNBT Western Blue stabilized substrate (Promega, Madison, WI) as described by the manufacturer. Radioiodination of IL-3 and binding assays. Radioiodination of E - 3 and binding assays were performed as previously described.” Briefly, low-affinity binding assays were performed by incubating 4 nmol/L lZ5I-IL-3with 5 X lo5 F6 cells at room temperature for 2.5 hours in RPMI 1640 containing 0.5% BSA and 0.1% sodium azide. After centrifuging through FCS, radioactivity in the cell pellet was determined by a Packard Auto-Gamma 5650 (Meriden, CT). When high-affinity binding assays were performed, 150 pmom I2’IIL-3 and 7 X 10’ COS cells co-expressing IL-3R a- and p-chains were used. In competition experiments, cells were incubated with ‘”1-IL-3 in the presence of a range of concentrations of MoAb 7G3 or IL-3. Radioiodination of MoAb 7G3 and binding assays. Ten micrograms MoAb 7G3 was iodinated with 0.5 mCi NalZSIby the Chloramine-T method.’” Saturation binding studies were performed by incubating 5 X lo5 F6 cells with Iz5I-7G3 over a range of concentrations (0.0018 to 20 nmol/L) in the presence or absence of a 100-fold excess of unlabeled MoAb 7G3. The binding curve was analyzed by Scatchard tran~formation.~’ In competition binding experiments, F6 cells or COS cells co-expressing IL-3R a- and pchains were preincubated for 2 hours at 4°C with a range of concentrations of IL-3 or GM-CSF before adding 1 nmol/L IZSI-7G3for another 2 hours. Inhibition of IL-3-mediated TF-I cell proliferation assay. TF1 cells were starved of GM-CSF for 24 to 48 hours before setting up proliferation assays. Briefly, 1 X lo4cells were incubated in wells with 0.3 nglmL IL-3 in the presence of a range of concentrations of MoAbs (0.00064 to 64 nmol/L) for 48 hours at 37°C. Wells were pulsed with 0.5 pCi per well 3H-thymidine for 4 hours and then harvested onto a glass filter, and radioactivities were determined by liquid scintillation and expressed as disintegrations per minute (DPM). IL-3-mediated histamine release from human basophils. Histamine release was determined as previously described.I4Briefly, lowdensity leukocytes were separated from peripheral blood by dextran sedimentation and centrifugation on Lymphoprep (Nycomed, Oslo, Norway). Cell suspensions (containing 1% to 2% basophils) were preincubated with purified human IgE for 45 minutes before incubating 1 x lo6 cells with E - 3 , a goat IgG anti-human IgE (0.8 pg/ mL), and a range of concentrations (O.OOO64 to 64 nmol/L) of MoAbs for a further 60 minutes. The released histamine was quantified subsequently using a radioenzymatic method.” IL-3-mediated functions on endothelial cells. The effect of MoAbs on IL-3-stimulated secretions of IL-6 and IL-8 by HUVEC From www.bloodjournal.org by guest on January 21, 2015. For personal use only. A NEUTRALIZING ANTI-IL-3Ra MoAb 85 B A 7G3 9F5 6H6 C kD C kD 763 9F5 6H6 > > 106 80 49.5 49.6 j j C * . :ri L. Q, P . E3 i *i i . . ! . z 0.1 1 10 100 lmo.l 1 10 100 1 m Monocytes Fig 1. MoAbs 763. 6H6, and 9F5 recognize t h e human IL-3R a-chain. (AI Immunoprecipitation of IL3R a-chain from '251-surface-labeled F6 cells and (B) Western blot of F6 cells. Both analyses were performed on 10% SDS-PAGE under reducing conditions. Molecular weight markers are shown to the left of each gel. (C) Flow cytometry analysis of stainings of MoAb 763 (-1 and the control MoAb (. . . to COS cells transiently transfected with IL3 R a-chain, F6 cells, neutrophils, monocytes, HUVEC, and eosinophils. d . nl -. was studied. HUVEC were obtained and cultured as previously described." For IL-6 measurements. HUVEC ( 5 x 10' per well) were treated with interferon gamma (IFN-7) (I00 UlmL) for 48 hours, IL-3 (30 nglmL) for 24 hours. or IFN-7 for 48 hours with IL-3 added for the last 24 hours with or without MoAhs 7G3 or 6H6 ( 100 pglmL). After treatment. the medium was changed and supernatants were collected for 24 hours and analyzed for the presence of immunoreactive IL-6 using an enzyme-linked immunosorbent assay (ELISA) method (Quantikine: R & D Systems, Minneapolis. MN). IL-8 production was measured as previously described.' Briefly, HUVEC (S X 10' per well) were incubated with tumor necrosis factor alpha (TNF-a) ( 100 UlmL) for 24 hours andlor IL-3 (30 ngl mL) for 6 hours with or without MoAb 7G3 (SO &mL). After incubation, the medium was changed and 1L-8 secreted in the following hour was quantified by ELISA. Cnnstnrcrion crnd erp'rs.sion of r/iitiwric N I I rrrmcarrd ~ IL-3R achtrins. The IL-3RalGM-CSFRa chimera is a fusion cDNA that encodes a chimeric receptor composed of the first 104 amino acids 1 io 100 iw Fluorescence Intensity of IL-3Ra including the signal sequence fused to amino acids I18 to 400 of the GM-CSFR a-chain. It was generated by polymerase chain reaction (PCR) using a sense primer 5' to the IL-3Ra coding sequence and an antisense primer corresponding to codons 104 to 99 and including a Kpn I site. The sequence of the resulting PCR product was checked, and it was then ligated in-frame to the Kpn I site at codon I I8 of GM-CSFRa. The GM-CSFRafL-3Ra chimera is a fusion cDNA that is the converse of the IL-3RalGM-CSFRa chimera and encodes the first 1 18 amino acids of GM-CSFR a-chain including the signal sequence fused to amino acids 104 to 378 of IL-3Ra. It was generated by PCR using a sense primer corresponding to codons 104 to I IO of IL-3Ra and includes a Kpn I site. A downstream antisense primer was also used. The resulting PCR product was sequenced and ligated in-frame to the Kpn I site at codon I I8 of GM-CSFRa. The IL-3Ra (-3 I ) flag is a cDNA that encodes an N-terminally truncated form of the IL-3Ra that lacks the first 31 amino acids of the mature protein but includes an eight amino acid "flag" peptide From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 86 SUN ET AL 3000 h 2500 2 a E 6000 a 5M: 2000 m 1500 el - i $ 1000 N 2000 500 0 0 10-2 10' 100 [MoAb] (nM) lo2 101 0 IO-' lo3 loo 10' [MoAb] (nM) sequence between the putative signal sequence and residue 50 of IL-3Ra. This cDNA was generated by digesting wild-type IL-3Ra with the restriction endonuclease EcoRV (Boehringer), which cleaves between codons 49 and 50, and ligating it to a PCR-generated fragment encoding the 18-amino acid signal sequence of the IL3Ra, the flag sequence, and a short multicloning sequence that results in Val-Asp-Asp separating the flag peptide and IL-3Ra. PCRgenerated sequences were verified by DNA sequence analysis. The IL-3Ra flag is a cDNA that encodes an IL-3Ra in which the putative signal sequence of IL-3Ra (first 18 amino acids) is fused to the flag peptide. It was generated by PCR using an upstream sense primer corresponding to codons 19 to 26 and carrying an Xhu I site at the 5' terminus of the primer. The downstream antisense primer corresponded to codons 104 to 99. The resultant PCR product was ligated at the 3' end to IL-3Ra (-31) flag using a common BamHI site to restore the coding sequence for the N-terminal 3 1 amino acids missing from IL-3Ra (-31) flag. The 5' end of the PCR product was ligated via the Xba I site to the 3' end of a PCR-generated fragment encoding the IL-3Ra signal peptide followed by the flag sequence plus the extra amino acids, Val-Asp-Asp-Ile-Ser-Arg. Fidelity of the PCR-generated portion was verified by DNA sequence analysis. All chimera and truncation constructs were cloned into the expression vector PMX139 before transfection into COS cells by DEAEdextran. Cells were grown to approximately 508 to 70% confluence, washed free of medium, and then incubated with 3 pg cDNA (per IO-cm plate) or 6 pg cDNA (per 15-cm plate) with 0.25 mg/mL DEAE-dextran. After approximately 30 minutes, the DEAE-dextran solution was aspirated and cells were washed and incubated in IMDM supplemented with 10% FCS and 100 pmol/L chloroquine for 3 to 5 hours. Finally, the cells were washed three times with rd Fig 2. Dose-dependent competition for '%IL-3 binding by MoAbs 763 (0),6H6 (0),9F5 (W), and a control MoAb (0) to (A) F6 CHO cells stably expressing IL3R a-chain and (61 COS cells transiently transfected with IL3R a - and p-chains. In A, '251-lL-3 was used at 4 nmol/L and in B at 150 pmol/L. I---)Competition by ZOO-fold-excess unlabeled IL3. Each point is the mean of t r i p licate determinations, and error bars represent standard deviations. serum-free medium and incubated with IMDM supplemented with 108 FCS for 40 to 60 hours at 37°C. RESULTS Development of MoAb 7G3. MoAb 7G3 and other antiIL-3Ra MoAbs, 6H6 and 9F5, were raised by immunizing mice with COS cell transfectants expressing the IL-3R achain on their surface and selecting on the stable CHO cell transfectant F6, which expresses 4 X 10' IL-3R a-chains per cell. MoAb 7G3, as well as MoAbs 6H6 and 9F5, bound strongly to F6 cells (Fig 1C) but not to untransfected CHO cells or CHO cell transfectants expressing GM-CSFR achain (data not shown). To confirm biochemically the identity of the antigen identified by MoAb 7G3 as the IL-3R achain, immunoprecipitation and Westem blot analysis were performed. MoAb 7G3, as well as MoAbs 9F5 and 6H6, specifically immunoprecipitated a protein of molecular weight 70,000 from '1 surface-labeled F6 cells, whereas a control anti-GM-CSFR a-chain MoAb failed to do so (Fig IA). MoAbs 7G3, 9F5, and 6H6 also recognized a protein of molecular weight 70,000 in Westem blotting of F6 cells (Fig 1B). No immunoprecipitated or Westem-blotted bands were seen when untransfected CHO cells were used (data not shown). Consistent with the known distribution of the IL-3R, MoAb 7G3 stained monocytes, HUVEC, and eosinophils but not fresh neutrophils (Fig lC), further confirming the identity of the antigen as the IL-3R a-chain. Identical staining was seen with 6H6 and 9F5 (data not shown). MoAb , 3000 . euw Fig 3. Dose-dependent competition for '%763 binding to (A) F6 cells stably expressing IL3R a-chain and (6) COS cells transiently transfected with IL3R (I- and p-chains by human IL-3 (0)or GM-CSF (0). I- -1 Inhibition in the presence of 100fold-excess unlabeled 7G3. Each point is the mean of triplicate determinations, and error bars represent standard deviations. - ui 5 8 5000 4000 --L -,,::,-,;-,.I-,; E 3001 & 2000 ,,,,,,,,,-, ,,,J 0 o loo 10' I$ io3 [Cytokine](nM) lo4 io5 53,,7 _ ,,,,,- - - - - -,,,,,,,, - - -,,,,,,,,- - , ,,,,,,,I 0 10-1 100 , , , 10' [Wokinel (nM) ld ,,l.l I d From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 87 A NEUTRALIZING ANTI-IL-3Ru MoAb 7G3 is classified as a mouse IgG2,, and 6H6 and 9F5 are mouse IgG, . Reciprocal inhibition of binding between IL-3 and MoAb 7G3. To examine whether the anti-IL-3R a-chain MoAb could interfere with IL-3 binding, we next performed competition experiments using lz5I-IL-3and cells expressing lowor high-affinity IL-3Rs. We found that MoAb 7G3 but not other MoAbs inhibited binding of Iz5I-IL-3to F6 cells expressing IL-3Ra in a dose-dependent manner (Fig 2A). Similarly MoAb 7G3 also blocked binding of Iz5I-IL-3to COS cells transfected with IL-3R a - and @-chaincDNA (Fig 2B). In both cases, MoAb 7G3 produced 50% inhibition of Iz5IIL-3 binding at approximately 0.7 nmoVL and complete inhibition at approximately 10 nmom. MoAbs 6H6 and 9F5 did not inhibit IL-3 binding to low- or high-affinity IL-3Rs; however, 6H6 enhanced Iz5I-IL-3binding to the IL-3R a chain (Fig 2A) in three of three experiments performed. In reciprocal competition experiments, F6 cells expressing the IL-3R a-chain alone (Fig 3A) or COS cells transfected with IL-3R a - and @chain cDNA (Fig 3B) were preincubated with IL-3 or GM-CSF over a range of concentrations before addition of '251-7G3.In both cases, IL-3 but not GM-CSF inhibited binding of Iz5I-7G3to the IL-3R. AfJinity of MoAb 7G3 for the IL-3R a-chain. Having established that MoAb 7G3 and IL-3 recognized the same or adjacent binding sites on IL-3R a-chain, we next performed direct measurements of MoAb 7G3 binding and compared them with IL-3 binding. Scatchard transformation of a saturation binding curve of IZ5I-7G3on F6 cells showed a kd of 900 pmol/L (Fig 4A). This represents a 100-fold higher affinity of IL-3Ra for 7G3 than reported for IL-3 itself.I6Consistent with these values, MoAb 7G3 competed with an approximately 100-fold greater affinity than IL-3 for Iz5I-IL-3 binding to F6 cells (Fig 4B). On the other hand, MoAb 7G3 competed with approximately threefold lower affinity than IL-3 on COS cells expressing the IL-3 high-affinity receptor (Fig 4C). MoAb 7G3 antagonizes IL-3-mediated biologic functions. Since IL-3 is a pleiotropic cytokine capable of stimulating multiple cell types and functions, we examined whether MoAb 7G3 could antagonize L - 3 functions in situations where IL-3 may play a pathogenic role, namely stimulation of cell proliferation, basophil histamine release, and endothelial cell activation. To study effects on proliferation, we used the TF-1 cell line, which is dependent on IL-3 for growth. A dose-response study indicated that a concentration of approximately 0.3 ng/mL IL-3 stimulated half-maximal proliferation of TF-1 cells (Fig 5A). We found that addition of MoAb 7G3 but not other anti-IL-3R a-chain MoAbs to TF-1 cells stimulated with 0.3 ng/mL IL-3 antagonized cell proliferation in a dose-dependent manner (Fig 5B). IL-3 has been shown to be one of the strongest enhancers of histamine release from human basophils, suggesting an effector role in all erg^.^,'^ From a dose-response of IL-3 (Fig 6A), we selected a concentration of 1 ng/mL to examine the effect of MoAb 7G3. We found that MoAb 7G3 but not MoAb 9F5 was able to completely antagonize IL-3-dependent stimulation of basophil histamine release (Fig 6B). Human endothelial cells have recently been shown to ex- A [1251-7G3bound] (nM) B C o IO* 10-i loo 10' 13 lo3 [Protein] (nM) Fig 4. Characterizationof MoAb lZ51-763binding to the IL-3R. (A) Scatchard transformationof a saturation binding curve using '"1-763 on F6 cells stably expressing IL-3R a-chain. (B)Competition for '"IIL-3 binding to F6 cells expressing IL-3R a-chain by MoAb 7 6 3 (0)or IL-3 (0). (C) Competition for 'z51-IL-3binding to COS cells expressing IL-3R a- and @chains by MoAb 7 6 3 (0)or IL-3 IO). Each point is the mean of triplicate determinations. press IL-3R a - and and it has been demonstrated that IL-3 acts as an amplification factor enhancing several endothelial cell functions, including cytokine secretion.' We found that MoAb 7G3 was able to antagonize the synergy of IL-3 with IFN-y in the stimulation of IL-6 secretion. This effect was specific for the IL-3 amplification effect and did not affect the small stimulatory effect of IFN-y alone (Fig 7A). Similarly, MoAb 7G3 was able to antagonize the en- From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 88 s 8 141 Y SUN ET AL n e 70000 i"1 3 6oooO Eg 50000 L - ,,,,,),,I ,,,,,,,,, g I C , y loo00 O , o ~ ld 10.2 W A b I (nW hancing effect of IL-3 on IL-8 secretion by TNF-a-stimulated cells without inhibiting the effect of TNF-a (Fig 7B). Epitope mapping of MoAb 7G3. To identify the region(s) in IL-3Ra recognized by MoAb 7G3, we initially tested MoAb 7G3 for binding to overlapping peptides of 14 amino acids in length encompassing the full extracellular domain of the IL-3R a-chain. However, no specific binding of MoAb 7G3 was observed (data not shown). Since these results suggest that MoAb 7G3 may recognize a conformational rather than a linear epitope, we generated cDNAs encoding IL-3RdGM-CSFRa chimeras and truncated IL3R a-chains (Fig 8A). These cDNAs were expressed in COS cells, and binding of MoAb 7G3 to the mutant receptors was examined by Western blotting and immunofluorescence. Although the IL-3Ra/GM-CSFRa chimera composed of amino acids 1 to 104 of IL-3Ra and amino acids 118 to 400 of GM-CSFRa bound 7G3 by both Western blot analysis (Fig 8B) and immunofluorescent microscopy (data not shown), the converse chimera (GM-CSFRa/IL-3Ra) composed of amino acids 1 to 118 of GM-CSFRa and amino acids 104 to 378 of IL-3Ra failed to do so. This suggests that the epitope for 7G3 is located in the amino-terminal 104 amino acids of IL-3Ra. A receptor deletion mutant, IL-3Ra (-31) flag, lacking the first 31 residues beyond the signal peptide (Thr 19-Asp 49 absent) but containing an eightresidue flag sequence, also failed to bind 7G3. However, another receptor mutant, IL-3Ra flag, containing Thr 19Asp 49 along with the flag sequence did bind 7G3 (Fig 8B). Fig 6. Inhibition of IL-3-mediated stimulation of human basophil histamine release by MoAb 763. (A) Histamine release in response to a range of concentrations of IL-3. (B) Histamine release stimulated by 1 ng/ mL IL-3 in the presence of a range of concentrations of MoAb 763 (O),9F5 (=I, and the control MoAb (0). Each value is the mean of quadruplicate determinations, and error bars represent standard deviations. Fig 5. Inhibition of IL-3-mediated proliferation of TF-1 cells by MoAb 763. (A) TF-1 cell proliferation in response to dflerent concentrations of IL-3. (B) TF-1 cell proliferation stimulated by 0.3 ng/mL IL-3 in the presence of a range of concentrations of MoAb 763 (O),6H6 (01.9F5 (Wl, and a control MoAb (0). Each pointisthemeanoftriplicatedeterminations, and error bars represent standard deviations. Strong expression of the IL-3Ra (-31) flag and IL-3Ra flag could be demonstrated by immunofluorescent microscopy (data not shown) and Westem blotting (Fig 8C) using an anti-flag M2 MoAb. These results suggest that the epitope of 7G3 may be located within amino acids Thr 19-Asp 49 of the amino terminus of IL-3Ra. DISCUSSION We describe here the generation of a specific anti-IL3Ra MoAb, 7G3, which completely and reciprocally inhibits the binding of IL-3 to its high- and low-affinity receptors and also antagonizes IL-3 activity in all functions tested. In addition, we show that the epitope recognized by MoAb 7G3 lies within the N-terminal domain of IL-3Ra, implicating this N-terminal domain, conserved among the IL-3Ra, GMCSFRa, and IL-5Ra subfamily, in ligand binding. MoAb 7G3 was one of a panel of MoAbs produced against hIL-3R a-chain. A single MoAb to hIL-3R a-chain has been previously described,35which recognizes subpopulations of peripheral blood and bone marrow cells. Using purified cell preparations, we show here that our MoAbs recognize human primary monocytes, eosinophils, and HUVEC, which by radioligand studies have been shown to express the IL-3R.5,7.'h On the other hand, the MoAbs do not stain neutrophils that do not bind 1L-336unless stimulated by GM-CSF.37These MoAbs recognize the IL-3R a-chain expressed on the cell surface by immunofluorescence, as well as either native or denatured IL-3Ra by immunoprecipitation or immunoblot. o 10.3 IO" i o 1 ioo [MoAb](nY) 10' id From www.bloodjournal.org by guest on January 21, 2015. For personal use only. A NEUTRALIZING ANTI-IL-3Ra MoAb 89 '" LB A h Fig 7. MoAb 7G3 selectively inhibits IL-3-mediated stimulation of (A) 11-6 release and (B) 1L8 release from HUVEC stimulated by 1L-3 (30 nglmL) together with IFN-y (100 UlmL) or TNF-a (100 U/mL). MoAb 7G3 was used at 30 pg/mL. Each value is the mean of triplicate determinations, and error b a n represent standard deviations. 1.B 6 m 5 4 =! NIL 11-3 IFN? IFNIL-3 These MoAbs are likely to be useful tools with which to study IL-3R expression and function. IL-3 is believed to play important roles in both hematopoiesis and inflammation. Although IL-3 has been shown to stimulate several cell types in vitro3' it is puzzling that this cytokine has not been detected in bone marrow or serum of normal animals,3g suggesting that it is not required for basal hematopoiesis. On the other hand, injection of IL-3 to mice and humans stimulates hematopoiesis, as well as causing significant side effects such as bone marrow fibrosis.'"," In this respect, IL-3 may be viewed as a "reactive" rather than a "steady-state" cytokine, and its production may lead to desirable, as well as potentially deleterious, effects. Consistent with this role, production of IL-3 is under tight regulatory control in T cells.42 We show here that MoAb 7G3 is an effective antagonist of IL-3 activities, with an EDSO of 0.4 to 1 nmol/L, consistent with its kd value (Fig 4A). Three types of IL-3 functions were studied since antagonism of IL-3 in these situations is likely to be of clinical significance. First, MoAb 7G3 antagonized IL-3-mediated enhancement of histamine release from basophils (Fig 6). Antagonizing IL-3 may be useful in allergic situations, since elevated IL-3 mRNA has been noted in the skin and bronchi of atopic individual^,'^ and the presence of IL-3 may lead to excessive stimulation of basophils and eosinophils at allergic reaction sites. Second, IL-3-mediated proliferation of the leukemic cell line, TF-1, was completely antagonized by MoAb 7G3 (Fig 5 ) at concentrations similar to those described earlier. Antagonism of IL-3-mediated cell proliferation is likely to be useful in some leukemias where IL-3 has been shown to promote growth.'"." In particular, follicular B-cell lymphomas, which bind IL-3 with high affinity and proliferate in an IL-3-dependent manner," may be ideally suited for intervention with MoAb 7G3. Finally, we found that MoAb 7G3 antagonized IL-3-mediated functions on HUVEC, namely enhancement of TNF-a stimulation and synergism with IFN-y (Fig 7). The presence of IL-3Rs on HUVEC and their upregulation by TNF-a and IFN-y has recently been n ~ t e d , ~ .and '.~~ their stimulation by IL-3 enhances IL-8 and IL-6 production, HLA class I1 expression,' and neutrophil tran~migration.~ Although the full IFNIL-3+ 7G3 IFNV 1L-3+ 6H6 NIL 11-3 TNF-a TNF-a+ TNF-(r+ TNF-a IL-3 1L-h 7G3 7G3 significance of these in vitro findings needs to be ascertained, these effects are likely to contribute to a systemic phase of inflammation and may be amenable to control with MoAb 7G3. MoAb 7G3 bound IL-3Ra with an approximate kd of 900 pmol/L. Thus, the affinity of MoAb 7G3 is approximately 100- to 300-fold greater than that of IL-3 for IL3Ra (100 nmol/L") and about 3- to IO-fold lower than that of IL-3 for the IL-3RaP high-affinity receptor (100 pmol/L"). This was reflected in the inhibition of IL-3 binding (Fig 4)and in the EDjo of MoAb 7G3 in functional assays (Figs 5 to 7). In other experiments, we have found that MoAb 7G3 prevents IL-3RaP heterodimerization (Stomski et al. in preparation) triggered by IL-3. This is consistent with a model in which IL-3 binding to IL-3Ra is required for receptor dimerization, and this is in turn essential for receptor activation. In competition experiments, we found that MoAb 7G3 and IL-3 reciprocally inhibited each other's binding. This suggests that the IL-3 binding site may lie within or adjacent to the epitope recognized by MoAb 7G3. In an effort to identify this epitope, which could also give clues as to the IL-3 binding sites, we initially used overlapping 14amino acid peptides spanning the whole extracellular domain of IL-3Ra. Since no specific reactivity was found, we then turned to truncated and chimeric receptors expressed on the surface of COS cells. We identified the N-terminal domain of IL-3Ra as a region required for MoAb 7G3 binding based on the positive immunofluorescence and Western blotting results with a chimeric receptor comprising the N-terminal domain of IL-3Ra and the CRM" of GM-CSFRa. In contrast, MoAb 7G3 failed to bind to a chimeric receptor comprising the N-terminal domain of GM-CSFRa and the CRM of the extracellular region of IL-3Ra (Fig 8). This suggests that the N-terminal domain of IL-3Ra is necessary for MoAb 7G3 binding. Further truncations in the Nterminus with retention of MoAb 7G3 reactivity suggest that the amino acid 19 to 49 region of the N-terminal domain of IL-3Ra forms part of the epitope recognized by MoAb 7G3. In other experiments (Barry et al, in preparation), we have found that truncation of the N-terminal domain of IL-3Ra does not abolish binding of From www.bloodjournal.org by guest on January 21, 2015. For personal use only. SUN ET AL 90 x 11AR CI FLAG LSXWS IY cc I L 3 R CI (-31) FLAG c <- 37n CD I LSXWS SO B kD --- <. 139 < 139 f- 84 84 Iy a < 42 MoAb 7G3 I -< 42 Anti-flag MoAb M2 IL-3, although the affinity of this binding is much decreased. These results have implications for defining the binding site for IL-3 and suggest that this may be formed by two noncontiguous regions in the primary structure of IL-3Ra, one of which is in the N-terminal domain and is recognized by MoAb 7G3. The existence of a conformational epitope for IL-3 and MoAb 7G3 is further supported by the inability of MoAb 7G3 to bind linear sequences as represented by the overlapping 14-amino acid peptides. It is interesting that the N-terminal domains of IL-3Ra, GM-CSFRa, and IL-5Ra represent a unique feature of this subfamily of receptors. They are not classic Ig-like domains and differ from the two domains of the cytokine receptor module encompassing the rest of each a-chain." Their function is not known, although the presence of apparently free Cys residues suggests a role in disulfidelinked heterodimerization with &chain (Stomski et al, in 1 378 Fig 8. (A) Schematic representation of IL-3% constructs used t o epitope-map MoAb 7G3. SP, signal peptide; TM, transmembrane region; CD, cytoplasmic domain. The conserved cysteines (c) and WSXWS motifs are indicated. Numbering of the primary sequence includes the signal peptide. Shaded regions represent GM-CSFR n-chain, and clear regions are IL-3R a-chain encoding DNA. (B) Western blot analysis of COS cells transiently transfected with various IL-3Ra mutants. Binding of 7G3 was seen with the IL-3RalGM-CSFRa chimera (B, lane 1) and with the wild-type lL-3Rn containing a flag sequence interposed between the signal peptide and residue 19, IL-3Ra flag (B, lane 41, but not with the GM-CSFRaI IL-3Ra chimera (B, lane 2) or with a truncated IL-3Ra lacking Thr 19-Asp 49, IL-3Ra (-31) flag (B, lane 3). (C) Expression of IL3Ra (-31) flag (C, lane 1) and IL3Ra flag (C, lane 2) are demonstrated by Western Blot using an anti-flag MoAb M2. preparation). The results shown here with IL-3Ra raise the possibility that the N-terminal domain of this subfamily of receptors is involved in ligand binding. ACKNOWLEDGMENT We thank B. Cambaren, M. Dottore, and M. Parsons for excellent technical assistance, and Man Walker for excellent secretarial assistance. REFERENCES I . Clark SC, Kamen R: The human hematopoietic colony-stimulating factors. Science 236: 1229, 1987 2. Sieff CA, Niemeyer CM, Nathan DG, Ekem SC, Bieber FR, Yang YC, Wong G, Clark SC: Stimulation of human hematopoietic colony formation by recombinant Gibbon-multi-colony-stimulating factor or interleukin 3. J Clin Invest 80:818. 1987 3. Lopez AF, Dyson PG, To LB. Elliott MJ. Milton SE, Russell From www.bloodjournal.org by guest on January 21, 2015. For personal use only. A NEUTRALIZING ANTI-IL-~RcTMoAb JA, Juttner CA, Yang Y, Clark SC, Vadas MA: Recombinant human interleukin-3 stimulation of hematopoiesis in humans: Loss of responsiveness with differentiation in the neutrophilic myeloid series. Blood 72:1797, 1988 4. Haak-Frendscho M, Arai N, Arai K-I, Baeza ML, Finn A, Kaplan AP: Human recombinant granulocyte-macrophage colonystimulating factor and interleukin 3 cause basophil histamine release. J Clin Invest 82:17, 1988 5. Elliott MJ, Vadas MA, Eglinton M, Park LS, To LB, Cleland LG, Clark SC, Lopez AF: Recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor show common biological effects and binding characteristics on human monocytes. Blood 74:2349, 1989 6. Saeland S, Duvert V, Moreau I, Banchereau J: Human B cell precursors proliferate and express CD23 after CD40 ligation. J Exp Med 178:113, 1993 7. Korpelainen EI, Gamble JR, Smith WB, Goodall GJ, Sun Q, Woodcock JM, Dottore M, Vadas MA, Lopez A F The receptor for interleukin-3 is selectively induced in human endothelial cells by tumour necrosis factor-a and potentiates interleukin-8 secretion and neutrophil transmigration. Proc Natl Acad Sci USA 90:11137, 1993 8. Korpelainen E, Gamble JR, Smith WB, Dottore M, Vadas MA, Lopez AF: Interferon-? upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex (MHC) class I1 expression and cytokine production. Blood 87:176, 1995 9. Orazi A, Cattoretti G, Schiro R, Siena S, Bregni M, Di Nicola M, Gianni AM: Recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor administered in vivo after high-dose cyclophosphamide cancer chemotherapy: Effect on hematopoiesis and microenvironment in human bone marrow. Blood 79:2610, 1992 10. Deliwel R, Salem M, Pellins C, Dorssers L, Wagemaker G, Clark S, Lowenberg B: Growth regulation of human acute leukemia: Effects of five recombinant hematopoietic factors in a serum-free culture system. Blood 72:1944, 1988 11. Park LS, Waldron PE, Friend D, Sassenfeld HM, Price V, Anderson A, Coman D, Andrews RG, Bemstein ID, Urdal DL: Interleukin-3, GM-CSF and G-CSF receptor expression on cell lines and primary leukemia cells: Heterogeneity and relationship to growth factor responsiveness. Blood 74:56, 1989 12. Clayberger C, Lune-Fineman S, Lee JE,Pillai A, Campbell M, Levy R, Krensky AM: Interleukin 3 is a growth factor for human follicular B cell lymphoma. J Exp Med 175:371, 1992 13. Valent P, Schmidt G, Besemer J, Mayer P, Zenke G, Liehl E, Hintgerger W, Lechner K, Maurer D, Bettelheim P: Interleukin-3 is a differentiation factor for human basophils. Blood 73:1763, 1989 14. Lopez AF, Eglinton JM, Lyons AB, Tapley PM, To LB, Park LS, Clark SC, Vadas MA: Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity. J Cell Physiol 145:69, 1990 15. Kay AB, Ying S, Varney V, Gaga M, Durham SR, Moqbel R, Wardlaw AJ, Hamid Q: Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/ macrophage colony-stimulating factor, in allergen-induced latephase cutaneous reactions in atopic subjects. J Exp Med 173:775, 1991 16. Kitamura T, Sat0 N, Arai K-I, Miyajima A: Expression cloning of the human IL-3 receptor cDNA reveals a shared P subunit for the human IL-3 and GM-CSF receptors. Cell 66:1165, 1991 17. Hayashida K, Kitamura T, Gorman DM, Arai K-I, Yokota T, Miyajima A: Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM- 91 CSF): Reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci USA 87:9655, 1990 18. Kitamura T, Miyajima A: Functional reconstitution of the human interleukin-3 receptor. Blood 80:84, 1992 19. Ihle JN, Witthuhn BA, Quelle FW,Yamamoto K, Thierfelder WE, Kreider B, Silvennoinen 0: Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci 19:222, 1994 20. Bazan JF:Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87:6934, 1990 21. Goodall GJ, Bagley CJ, Vadas MA, Lopez AF: A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands. Growth Factors 8:87, 1993 22. Hercus TR, Camhareri B, Dottore M, Woodcock J, Bagley CJ, Vadas MA, Shannon MF, Lopez AF: Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the a- and P-chains of its receptor. Blood 83:3500, 1994 23. Woodcock JM, Zacharakis B, Plaetinck G, Bagley CJ, Qiyu S, Hercus TR, Tavemier J, Lopez AF: Three residues in the common chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu2' of GM-CSF. EMBO J 13:5176, 1994 24. Barry SC, Bagley CJ, Phillips J, Dottore M, Cambareri B, Moretti P, D'Andrea R, Goodall GJ, Shannon MF, Vadas MA, Lopez AF: Two contiguous residues in human interleukin-3, Asp2' and G1uZ2,selectively interact with the a- and P-chains of its receptor and participate in function. J Biol Chem 269:8488, 1994 25. Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC: Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550, 1977 26. Rathjen DA, Geczy CL: Conditional medium from macrophage cell lines supports the single-cell growth of hybridomas. Hybridoma 5:255, 1986 27. Lyons AB, Ashman LK: The Rose Bengal assay for monoclonal antibodies to cell surface antigens: Comparisons with common hybridoma screening methods. J Immunoassay 6:325, 1985 28. Krissansen GF, Lucas CM, Stomski FC, Elliott MJ, Bemdt MC, Boyd AW, Horton MA, Cheresh DA, Vadas MA, Burns GF: Blood leukocytes bind platelet glycoprotein (IIb-Ma)' but do not express the vitronectin receptor. Int Immunol 2:267, 1989 29. Lopez AF, Eglinton JM, Gillis D, Park LS, Clark S, Vadas MA: Reciprocal inhibition of binding between interleukin 3 and granulocyte-macrophage colony-stimulating factor to human eosinophils. Proc Natl Acad Sci USA 86:7022, 1989 30. McConahey PJ, Dixon FJ: Radioiodination of proteins by the use of the chloramine-T method. Methods Enzymol 70A:210, 1980 31. Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949 32. Shaff RE, Beavan MA: Increased sensitivity of the enzymatic isotopic assays of histamine: Measurement of histamine in plasma and serum. Anal Biochem 94:425, 1979 33. Gamble JR, Elliott MJ, Jaipargas E, Lopez AF, Vadas MA: Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci USA 86:7169, 1989 34. Brizzi MF, Garbarino G, Rossi PR, Pagliardi GL, Arduino C, Avanzi GC, Pegoraro L: Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells. J Clin Invest 91:2887, 1993 35. Sat0 N, Caux C, Kitamura T, Watanabe Y, Arai K-I, Banchereau J, Miyajima A: Expression and facter-dependent modulation of the interleukin-3 receptor subunits on human hematopoietic cells. Blood 82:752, 1993 36. Lopez AF, Eglinton JM, Gillis D, Park LS, Clark S, Vadas MA: Reciprocal inhibition of binding between interleukin 3 and From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 92 granulocyte-macrophagecolony-stimulating factor to human eosinophils. Proc Natl Acad Sci USA 86:7022, 1989 37. Smith WB, Guida L, Sun Q,Korpelainen EI, Gillis D, van den Heuvel C, Hawrylowiez CM, Vadas MA, Lopez AF: Neutrophils activated by GM-CSF express receptor for interleukin-3 which mediate class I1 expression. Blood (in press) 38. Metcalf D: The Hemopoietic Colony Stimulating Factors. Amsterdam, The Netherlands, Elsevier, 1984, p 493 39. Metcalf D: Control of granulocytes and macrophages: Molecular, cellular, and clinical aspects. Science 254529, 1991 SUN ET AL 40. Metcalf D, Begley CG, Johnson GR, Nicola NA, Lopez AF, Williamson DJ: Effects of purified bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice. Blood 68:46, 1986 41. Falk S , Seipelt G, Ganser A, Ottmann OG, Hoelzer D, Stutte HJ, Hubner K Bone marrow findings after treatment with recombinant human interleukin-3. Am J Clin Pathol 9S:3SS, 1991 42. Ryan GR, Vadas MA, Shannon MF: T-cell functional regions of the human IL-3 proximal promoter. Mol Reprod Dev 39:200, 1994 From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 1996 87: 83-92 Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist Q Sun, JM Woodcock, A Rapoport, FC Stomski, EI Korpelainen, CJ Bagley, GJ Goodall, WB Smith, JR Gamble, MA Vadas and AF Lopez Updated information and services can be found at: http://www.bloodjournal.org/content/87/1/83.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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