By
From the * Cell Biology Unit, GlaxoWellcome Research and Development, Medicines Research
Centre, Stevenage, Hertfordshire, SG1 2NY, United Kingdom; and the Geneva Biomedical
Research Institute, GlaxoWellcome Research and Development S.A., Geneva, Switzerland
Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.
IL-5 is a cytokine secreted predominantly by T cells, but
also by mast cells and eosinophils (1), that has multiple
effects on cells of the eosinophil and basophil lineage. IL-5
induces the differentiation and expansion of eosinophil
precursors in the bone marrow (4) and plays a key role in
regulating many of the specialized functions of mature, terminally differentiated eosinophils, including adhesion (5),
priming of both degranulation and chemotaxis (6, 7), and
the promotion of cell survival (8).
While eosinophils appear to play a protective role during
the host defense response to parasitic infection, accumulating clinical and experimental evidence has also implicated
the eosinophil in the pathophysiology of a number of allergic diseases such as bronchial asthma, allergic rhinitis, and
atopic dermatitis (9). Administration of neutralizing anti-
IL-5 monoclonal antibodies in mouse, guinea pig, or primate models of allergic asthma inhibits the development of
airway eosinophilia and bronchial hyperreactivity (12).
This highlights the eosinophil, and more specifically IL-5,
as an attractive target for therapeutic intervention in allergic
diseases such as asthma.
The effects of IL-5 are mediated through a heterodimeric receptor complex composed of a specific ligand binding IL-5 is a disulphide-linked homodimeric glycoprotein with
115 amino acids in each chain (26). Elucidation of the IL-5
crystal structure revealed a novel two domain structure in
which each domain adopts a four In this study we have produced an IL-5 mutant containing a charge reversal at position 12, IL-5 (E12K). It has
been characterized in a variety of functional and receptor
binding assays, and we have used this protein as a tool to
reveal distinct mechanisms of receptor/cellular activation in
human eosinophils.
Reagents.
Recombinant IL-3, TNF- Site-directed Mutagenesis and Protein Expression.
Recombinant
human IL-5 was expressed in E. coli and purified to homogeneity
as previously described (30). IL-5 (E12K) was generated by site-directed mutagenesis, expressed in E. coli and purified as previously described (28) with the following modifications. Renaturation was carried out by dropwise dilution of the protein in 6 M
urea, into 0.1 M ethanolamine, pH 9.8, containing 2.5 M urea,
10% ammonium sulphate, 1 mM reduced glutathione, and 0.1 mM
oxidized glutathione to a final protein concentration of 10 µg/ml,
and stirred overnight at 4°C. The solution was dialyzed against
three changes of 0.1 M ethanolamine, and concentrated 10-fold
by ultrafiltration before gel filtration. Circular dichroism spectral
analysis confirmed that the secondary and tertiary structure was
identical to wild-type IL-5 (data not shown).
Receptor Binding Assays.
In competition binding studies IL-5
and IL-5 (E12K) were assayed for their relative ability to bind to recombinant IL-5 receptor subunit and a
subunit that is shared by the specific
receptor
chains for IL-3 and GM-CSF (17). Although
this common
chain (
c) alone has no inherent ligand
binding capacity, it confers high-affinity ligand binding to
the receptor
/
chain complex. This phenomenon of increased binding affinity in the presence of the
c chain has
been termed affinity conversion, and is most profound for IL-3 (1,000-fold), with smaller effects on GM-CSF binding
(20-100-fold) and IL-5 binding (2-4-fold) (18). The
roles of the respective
and
c chains in signal transduction are unclear. Analysis of chimeric receptors composed
of the extracellular domain of the IL-5 receptor
chain
and the intracellular domain of the
c chain suggests that
ligand-induced dimerization of the cytoplasmic domains of
c may be sufficient for receptor activation and the transduction of a proliferative signal (21). In contrast, deletion
and mutational analysis has defined specific regions within
the cytoplasmic domains of both the IL-5 receptor
and
c chains that are essential for the coupling of IL-5 receptors to a proliferative signal (21). Thus, although
c is
indispensable for the signals leading to proliferation, it is
possible that the receptor
chain may contribute to cytokine signal transduction either directly, or by promoting receptor oligomerization. Furthermore, the relative contributions of the two receptor chains to signal transduction
leading to functional responses other than proliferation are
uncharacterized. Although the precise molecular mechanisms by which the active IL-5 receptor complex directs
these diverse cellular responses in the eosinophil are also
poorly understood, one of the earliest measurable signaling events is the phosphorylation of a variety of cellular proteins on tyrosine residues (25).
-helix bundle structure
similar to the cytokine fold in IL-2, IL-4, growth hormone, and GM-CSF (27). Among these related structures,
IL-5 is unique in that each bundle is composed of three helices from one monomer and a fourth helix which is contributed by the second monomer. Recently, the receptor
binding sites on IL-5 have been defined by site-directed
mutagenesis. Residues E88, R90, and E109, within the
carboxy terminus of IL-5, define the IL-5 receptor
chain
binding site (28, 29). In addition, the IL-5 mutant E12A,
IL-5 (E12A), exhibits partial agonist activity in a TF-1 cell
proliferation assay, suggesting that the highly conserved E12
in the amino-terminal helix defines a point of contact with
the receptor
c chain which is important for
c chain activation and subsequent signal transduction (28). Furthermore, in a TF-1 proliferation assay the IL-5 mutant E13Q (Due to a single amino acid difference at the amino terminus, IL-5 [E12] as described here is equivalent to IL-5 [E13]
as described by Tavernier [29].), IL-5 (E13Q), was completely inactive and exhibited antagonist properties (29).
, and GM-CSF were
obtained from R&D systems (Abingdon, UK). BS3 (bis[sulfosuccinimidyl] suberate)1 cross-linking reagent was from Pierce and
Warriner (Chester, UK). Na125I for protein iodination, and 125I-labeled interleukin 5 (125I-IL-5) were obtained from Amersham
International (Amersham, Buckinghamshire, UK). Anti-phosphotyrosine antibody (4G10) was obtained from TCS Biologicals
(Buckingham, UK). All other chemicals were obtained from
Sigma (Poole, Dorset, UK).
chain alone, or the receptor
/
chain
complex as expressed on TF-1 cells, as previously described (31).
chain, fused with the IgG binding domain of protein A (IL-5R
-ZZ), was incubated with rabbit IgG and anti-rabbit-coated fluoromicrosphere beads (Amersham International, UK) for 2 h at 4°C. The resultant complex was incubated with 100 pM 125I-IL-5 in the presence of increasing concentrations of unlabeled cytokines as indicated. After incubation for
4 h at room temperature, bound radioligand was measured in a
Wallac 1450 Microbeta scintillation counter set up in scintillation
proximity assay (SPA) mode. Nonspecific binding was measured
in the presence of a 500-fold molar excess of unlabeled IL-5.
TF-1 Cell Proliferation Assay. Cytokine-induced proliferation of the human erythroleukemia cell line TF-1 was measured as previously described (28). In brief, assays were performed in 96-well microtiter plates containing 5 × 103 cells/well with the indicated cytokines. After incubation for 60-72 h at 37°C the induction of proliferation was measured using the Cell Titer 96TM nonradioactive cell proliferation assay (Promega, Southampton, UK), according to the manufacturer's instructions.
Eosinophil Purification. Human peripheral blood eosinophils were isolated from healthy donors with mildly elevated eosinophil count, by a CD16-negative selection protocol as previously described (32). Eosinophil purity was always >95%.
Eosinophil Activation Assay. Cytokine-induced activation of purified eosinophils, as measured by adhesion to immobilized IgG, was assayed as previously described (32). In brief, 5 × 103 eosinophils were incubated with the indicated cytokines for 30 min at 37°C, in a 96-well microtiter plate precoated with human IgG. After a washing step, the adherent eosinophils were lysed and the endogenous peroxidase activity measured in a colorimetric assay using o-phenylenediamine as a substrate.
Eosinophil Survival Assay. Purified eosinophils were suspended at 106 cells/ml in DMEM containing 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin (GIBCO, Paisley, Scotland) in microtiter plates, in the presence or absence of wild-type IL-5 or IL-5 (E12K). After incubation at 37°C for 72 h, cell viability was assessed by trypan blue exclusion, counting a minimum of 200 cells. For antibody blocking experiments, human eosinophils were incubated with the indicated concentrations of cytokines in the presence or absence of 250 µg/ml anti-IL-5 neutralizing antibody, TRFK-5 (33). For polymyxin B experiments agonists were preincubated with 500 U/ml polymyxin B for 1 h at 37°C before addition of purified human eosinophils.
Cross-linking of Radiolabeled IL-5 or IL-5 (E12K) to IL-5 Receptor
and
Chains Expressed on COS Cells.
For cross-linking studies recombinant human IL-5 and IL-5 (E12K) were iodinated by
a modified chloramine-T method essentially as described previously (28).
Detection of Phosphotyrosine Containing Proteins by Western Blotting. Human eosinophils (5 × 105) were incubated in the presence of indicated concentrations of cytokines for 5 min at 37°C, in PBS containing 0.4% human serum albumin. The cells were then pelleted at 4°C, resuspended in 1× NuPAGE sample buffer containing 2.5% 2-ME and 1 mM sodium orthovanadate and boiled for 5 min. Extracts were analyzed on a 4-12% NuPAGE SDS-polyacrylamide gel run in MOPS buffer and transferred to hybond-ECL membrane (Amersham International, Amersham, UK). Blots were probed with the anti-phosphotyrosine antibody 4G10, and proteins visualized using a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Sigma, Poole, Dorset, UK) with enhanced chemiluminescence (ECL) detection (Amersham International, UK).
Data Analysis. Receptor binding and biological data were analyzed using Grafit 3.01 as previously described (28).
Wild-type human IL-5 and a mutant IL-5 containing a
charge reversal mutation at position 12, IL-5 (E12K), were
expressed in E. coli and purified to homogeneity. As a
means of comparing the receptor binding properties of wild-type IL-5 and IL-5 (E12K), both proteins were assayed for
their relative ability to bind to the IL-5 receptor chain
alone, or to the high-affinity
/
receptor complex. In
competition binding experiments, using the recombinant
extracellular domain of the IL-5 receptor
chain in an
SPA assay format, IL-5 (E12K) exhibited a 1.7 ± 0.2-fold (n = 6)-fold reduction in binding affinity to the receptor
chain relative to wild-type IL-5 (Fig. 1 A). The interaction
with the high-affinity receptor complex was assessed in a
cell based competition binding assay using TF-1 cells which
express both the IL-5 receptor
and
c chains. In this cell
based system IL-5 (E12K) exhibited a 4.5 ± 1.2-fold (n = 5)
reduction in binding affinity relative to wild-type IL-5
(Fig. 1 B). The shift in relative binding of IL-5 (E12K) and
wild-type IL-5 to the high-affinity
/
complex, compared
to the
chain alone, was found to be statistically nonsignificant (P = 0.084), as assessed by an analysis of variance
technique.
Chemical Cross-linking of Radiolabeled IL-5 and IL-5 (E12K) to IL-5 Receptors.
To determine whether IL-5 (E12K) is able
to interact with the IL-5 receptor c chain, we employed
receptor cross-linking studies. The chemical cross-linking
of 125I-IL-5 or 125I-IL-5 (E12K) to COS cells transfected
with IL-5 receptor
and
c chains revealed identical patterns of cross-linked species of ~70 and 150 kD under reducing conditions (Fig. 2), which correspond to the predicted molecular mass of the receptor
and
c subunits, respectively, bound to a monomer of IL-5. Furthermore,
the appearance of these bands was blocked by addition of a
100-fold molar excess of unlabeled IL-5 during the incubation, confirming that both 125I-IL-5 and 125I-IL-5 (E12K)
can be specifically cross-linked to both IL-5 receptor
and
c chains.
Biological Properties of IL-5 (E12K) in a TF-1 Proliferation Assay.
Wild-type IL-5 induced the proliferation of the
human erythroleukemia cell line TF-1 in a concentration-dependent fashion (ED50 = 2.6 ± 0.6 pM, n = 4), while
IL-5 (E12K) was unable to stimulate proliferation even at
concentrations up to 400 nM, a concentration 160,000-fold higher than the concentrations of wild-type IL-5 required to elicit half maximal proliferation of TF-1 cells (Fig. 3 A).
Having determined that IL-5 (E12K) was able to bind to IL-5 receptors with almost wild-type affinity, yet was unable to elicit a proliferative response in TF-1 cells, we tested this mutant for its ability to antagonize the effects of IL-5 in a TF-1 proliferation assay. As seen in Fig. 3 B, IL-5 (E12K) antagonized the effect of IL-5 in a concentration-dependent manner. Proliferation of TF-1 cells, in the presence of 77 pM wild-type IL-5, was totally inhibited by 100 nM IL-5 (E12K) and 50% inhibition was achieved at 10.3 ± 2.8 nM (n = 6), which represents a 130-fold molar excess of IL-5 (E12K) over wild-type. Furthermore, in the same functional assay IL-5 (E12K) did not significantly inhibit TF-1 proliferation stimulated by IL-3 or GM-CSF (Fig. 3 B). This demonstrates not only that IL-5 (E12K) is a specific IL-5 receptor antagonist, but also excludes the possibility of IL-5 (E12K)-dependent cellular toxicity.
Biological Properties of IL-5 (E12K) in an Eosinophil Activation Assay.To confirm and extend our observations to
additional IL-5-induced cellular responses, we switched
our studies to human peripheral blood eosinophils, a terminally differentiated cell type in which IL-5 stimulates multiple functional responses. We have established a sensitive and
reliable in vitro assay for analysis of cytokine-induced eosinophil activation based on adhesion to immobilized IgG. In this assay, wild-type IL-5 induced a concentration-dependent increase in eosinophil adhesion to IgG (ED50 = 1.3 ± 0.1 pM, n = 3), while the mutant E12K was incapable of
inducing eosinophil adhesion even up to concentrations of
1 µM, a concentration 400,000-fold higher than the ED50
for wild-type IL-5 (Fig. 4 A). In the same functional assay
IL-5 (E12K) completely antagonized the effects of IL-5 in a
concentration-dependent fashion. Eosinophil adhesion in the
presence of 20 pM wild-type IL-5 was completely inhibited by 50 nM IL-5 (E12K) with 50% inhibition achieved
at 1.9 ± 0.1 nM (n = 3), which represents a 95-fold molar
excess of IL-5 (E12K) over wild-type (Fig. 4 B). In addition, the inhibitory effect of IL-5 (E12K) was specific for
IL-5 induced adhesion with no significant inhibition of IL-3,
GM-CSF, or TNF- across the range of IL-5 (E12K) concentrations found to inhibit IL-5 (Fig. 4 B).
Biological Properties of IL-5 (E12K) in an Eosinophil Survival Assay.
Mature eosinophils that have been separated from
peripheral blood do not survive more than 4 d in vitro
without the addition of cytokines. The eosinophilopoietic
cytokines IL-5, IL-3, and GM-CSF have all been reported
to promote eosinophil survival, and so maintain cell viability (8, 34, 35). We therefore assessed the relative ability of
wild-type IL-5 and IL-5 (E12K) to promote eosinophil survival. Purified human peripheral blood eosinophils were
incubated in the presence of increasing concentrations of
wild-type IL-5 or IL-5 (E12K) and eosinophil viability
measured by trypan blue exclusion, after a period of 72 h.
Both IL-5 and IL-5 (E12K) were able to promote eosinophil survival in a concentration-dependent manner with
ED50 values of 0.4 ± 0.1 pM and 20.6 ± 8.7 nM, respectively (n = 5) (Fig. 5 A). Although IL-5 (E12K) promoted
eosinophil survival with a 50,000-fold reduction in biological potency with respect to wild-type IL-5, it was still a
full agonist capable of eliciting a maximal biological response not significantly different from wild-type IL-5.
One trivial explanation for the agonist activity of IL-5 (E12K) in the eosinophil survival assay is the contamination of this E. coli expressed cytokine with bacterial LPS, which has been reported to promote eosinophil survival through the autocrine production of GM-CSF (36). To exclude this possibility a series of control experiments were performed (Fig. 5 B). First, the agonist activity of IL-5 (E12K) was completely abolished after boiling for 15 min. Furthermore, pretreatment with polymyxin B, an LPS inhibitor (36), blocked LPS- but not IL-5 or IL-5 (E12K)-induced eosinophil survival. In addition, the anti-IL-5 neutralizing antibody TRFK-5 blocked the agonist activity of IL-5 (E12K) and wild-type IL-5, but not IL-3, GM-CSF or LPS in the eosinophil survival assay (Fig. 5 B). This confirms that the eosinophil survival activity is specifically mediated via IL-5 (E12K) and excludes the possibility of an LPS-dependent mechanism.
Relative Effects of IL-5 and IL-5 (E12K) on Tyrosine Phosphorylation in Eosinophils.Stimulation of eosinophils with
IL-5 induced an increase in the phosphotyrosine content of
a number of proteins, in a concentration-dependent manner, with the predominant phosphorylated species at 150-kD (Fig. 6). In contrast to wild-type IL-5, IL-5 (E12K) did not
stimulate tyrosine phosphorylation even at concentrations up
to 5 µM. Furthermore, 500 nM IL-5 (E12K) completely
inhibited the tyrosine phosphorylation of this 150-kD protein stimulated by 250 pM IL-5 (Fig. 6).
In a systematic program of alanine scanning mutagenesis
we previously demonstrated that IL-5 (E12A) exhibited
partial agonist activity in a TF-1 cell proliferation assay and
proposed that E12 defined a contact point for the IL-5 receptor chain. We based this conclusion not only on biological activity but on the reduction in affinity of E12A for
the IL-5 receptor
/
chain compared to the
chain alone
(28). Generating a charge reversal at this putative
c chain
binding site residue, IL-5 (E12K), has allowed us to further
investigate the effect of this mutation.
Glutamic acid-12 resides on the amino-terminal helix of
IL-5 at an analogous position to E21 in GM-CSF and E22
in IL-3. Charge reversal mutations at these positions in IL-3
and GM-CSF resulted in proteins which were unaltered in
binding their respective chains, but demonstrated a reduction in high-affinity binding (37, 38). For IL-5 (E12K)
we also see essentially wild-type binding to the IL-5 receptor
chain, but a reduction in high affinity binding of
~2.6-fold. Although this result is entirely consistent with
the reduction in high affinity we previously observed with
IL-5 (E12A) (28), the apparent reduction in the affinity of
IL-5 (E12K) for the
/
complex is not statistically significant (P = 0.084). Since the affinity conversion conferred
by
c on the IL-5/IL-5 receptor-
chain complex is small
(two- to fourfold), compared to the much larger shifts in
affinity seen with both GM-CSF and IL-3, this lack of statistical significance is probably due to the difficulty of measuring such small changes. The contribution of the
c chain
to IL-5 (E12K) high-affinity binding is difficult to measure
clearly through competition binding studies. However we
demonstrated by chemical cross-linking studies that, as with wild-type IL-5, IL-5 (E12K) is still capable of contacting
not only the receptor
chain but also the
c chain. This
result is consistent with the pattern of cross-linking obtained with IL-5 (E13Q), which has a similar antagonist biological profile in TF-1 cells (29). Such cross-linking experiments have not been published with human GM-CSF
(E22R) or IL-3 (E21R) and it is not clear whether these
mutants can similarly cross-link to the
c chain in the absence of high affinity binding. However, a biologically active murine GM-CSF mutant (E21A) has been described
which binds the
/
complex with low affinity only, yet is
able to cross-link to both the
and
c chains of the receptor (39). As suggested by Tavernier et al. (29), this may indicate that affinity conversion and receptor activation can
be uncoupled.
In contrast to IL-5 (E12A), the IL-5 (E12K) mutant was
completely devoid of agonist activity in a TF-1 cell proliferation assay and was a full and selective antagonist of IL-5
in this functional assay. While IL-3 (E21R) retains agonist
activity for TF-1 proliferation at 20,000-fold reduced potency relative to wild-type IL-3 (37), GM-CSF (E22R)
lacks agonist activity and is a specific GM-CSF antagonist
(40). Furthermore, the antagonistic properties of IL-5 (E12K)
are not restricted to inhibition of IL-5-induced cell proliferation. In human eosinophils IL-5 (E12K) was a selective
antagonist of IL-5 induced eosinophil activation, as assessed
by adhesion to immobilized IgG, exhibiting no effect on
eosinophil activation induced by the related cytokines IL-3 and GM-CSF or the unrelated cytokine TNF-.
Taken together these mutagenesis studies suggest that the
structural conservation at both the cytokine and receptor
level for IL-5, IL-3, and GM-CSF underlie a common
mechanism of receptor binding and activation, and that the
acidic residues within the amino terminal helix of all three
cytokines form a contact point for the c chain which contributes to receptor activation and, for the latter two cytokines at least, high-affinity binding. Somewhat surprisingly
however, IL-5 (E12K) was clearly a full agonist in an eosinophil survival assay, albeit with a 50,000-fold reduction in
potency relative to wild-type IL-5. This survival effect of
IL-5 (E12K) was not due to the presence of contaminating LPS and was consistently seen in the same eosinophils in
which IL-5 (E12K) antagonized IL-5-induced adhesion to
IgG. Clearly this single point mutation in IL-5 is able to
distinguish between two IL-5-dependent functional responses in the same cell type. This apparent functional dichotomy in the actions of IL-5 (E12K) implies that different
functional responses within the same cell, activation versus
survival, are mediated through distinct mechanisms of cellular activation.
In the context of IL-5, E12 appears to be necessary for
activating the IL-5 receptor c leading to an eosinophil activation signal. However, the fact that IL-5 (E12K) promotes eosinophil survival suggests that residues on IL-5
other than E12 are involved in receptor activation leading
to a survival signal. The reduction in potency of IL-5
(E12K) with respect to wild-type IL-5 further suggests that,
although not required for full agonist activity, E12 must
contribute to the efficiency of receptor activation. This
would indicate that multiple points of contact between IL-5 and its receptor complex are required for complete receptor activation. Since IL-5 (E12K) can be cross-linked to the
receptor
c, residues other than E12 may directly contact
c contributing to its complete activation. Such a model
may also account for the ability of human IL-3 (E22R) and
murine GM-CSF (E21A) to exhibit biological activity despite the lack of high-affinity binding (37,39). However, a direct role for the IL-5 receptor
chain in signal transduction has been inferred from both deletion and point mutation studies in the intracellular domain of the
chain, resulting in the loss of IL-5-induced cell proliferation (21,
22). Furthermore, it has been proposed that the ligand-specific
subunits of the IL-5, IL-3, and GM-CSF receptors
may mediate cytokine-specific signals leading to different
cellular responses (41). Since mutations at E12 appear to be
unaffected in
-chain binding, it is also possible that elements of the survival signal emanate from the
chain.
These properties of IL-5 (E12K) highlight its potential use as a tool for dissecting signaling pathways from the activated IL-5 receptor complex leading to distinct IL-5-dependent functional responses, in particular eosinophil survival. Initial experiments in eosinophils measuring one of the earliest detectable signaling events, tyrosine phosphorylation, highlighted opposing effects of IL-5 and IL-5 (E12K). Wild-type IL-5, but not IL-5 (E12K), was able to activate tyrosine phosphorylation of a major 150-kD protein species and furthermore, the IL-5-dependent phosphorylation of this protein was inhibited by IL-5 (E12K).
In summary, we have identified an IL-5 point mutant, IL-5 (E12K), which lacks agonist activity and is a potent antagonist in both a TF-1 cell proliferation assay and an eosinophil activation assay, yet exhibits agonist activity in promoting eosinophil survival. The dual agonist and antagonist properties of IL-5 (E12K) can be explained via distinct mechanisms of cellular/receptor activation. We are currently attempting to elucidate the precise mechanism by which this IL-5 mutant exerts its agonist effect.
Address correspondence to Dr. Murray McKinnon, Cell Biology Unit, GlaxoWellcome Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK. Phone: 01438-745745; FAX: 01438-763232.
Received for publication 30 January 1997 and in revised form 22 April 1997.
1 Abbreviations used in this paper: BS3, bis(sulfosuccinimidyl) suberate; ED80, concentration required to give 80% of the maximum biological response; IL-5R-zz, extracellular domain of the IL-5 receptorThe authors would like to thank Miss Gillian Amphlett for statistical analysis and Dr. Tim Mosmann (DNAX, Palo Alto, CA) for providing TRFK-5.
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