(Received for publication, September 11, 1996)
From the Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Physiologische Chemie II, Am Hubland, D-97074 Würzburg, Germany
We characterize here a highly efficient
antagonist for interleukin-4 (IL-4) in the mouse system. In this double
mutant of the murine IL-4 protein, both glutamine 116 and tyrosine 119 were substituted by aspartic acid residues. This variant (QY) bound with similar affinity to the IL-4 receptor subunit as wild type IL-4 without inducing cellular responses. In contrast, QY completely inhibited in a dose-dependent manner the IL-4-induced
proliferation of lipopolysaccharide-stimulated murine splenic B-cells,
of the murine T cell line CTLL-2, and of the murine pre-B-cell line
BA/F3. QY also inhibited the IL-4-stimulated up-regulation of CD23
expression by lipopolysaccharide-stimulated murine splenic B-cells and
abolished tyrosine phosphorylation of the transcription factor Stat6
and the tyrosine kinase Jak3 in IL-4-stimulated BA/F3 cells. Selective inhibition of IL-4 may be beneficial in T-helper cell type 2-dominated diseases, like type I hypersensitivity reactions or helminthic infections. The QY mutant could be an attractive tool to study in
vivo the therapeutic potential of IL-4 antagonists in mouse systems.
Interleukin-4 (IL-4)1 is a pleiotropic
cytokine derived from T-cells, thymocytes, and mast cells that has
multiple effects on many cell types (1). Its functions include the
differentiation of T helper cells to a TH2 phenotype and
the induction of sterile transcripts in B-cells, which is a
required step for class switching to IgE type antibodies (2, 3). Among
the clinically important features of IL-4 are the coordination of
immune responses against helminthic macroparasites and its central role
in the sensitization process of type I allergic diseases (4, 5).
Two receptor proteins have been identified for IL-4. The 140-kDa IL-4
receptor chain (IL-4R
) binds IL-4 with high affinity (6-8). The
second subunit is the 64-kDa common
receptor subunit (
c) (9,
10), which is also shared by the receptors for IL-2, IL-7, IL-9, and
IL-15 (11).
Site-directed mutagenesis has led to the discovery of two regions in
the human IL-4 molecule that are important for interaction with the
receptor chains (for review see Ref. 12). Substitution of glutamic acid
9 (Glu9) or arginine 88 (Arg88) leads to a near
complete loss of binding to IL-4R (13). A second important region of
the human IL-4 molecule is defined by mutations of arginine 121, tyrosine 124, and serine 125, close to the C terminus of the protein.
Mutations at these positions do not interfere with IL-4R
binding but
can severely impair cellular responses, because they result in loss of
interaction with
c (14). The most efficient single site mutant of
this type is Y124D, which does not induce T-cell proliferation but is a
partial agonist for up-regulation of CD23 on B-cells (15). In contrast, a double mutant where both Arg121 and Tyr124
have been replaced by aspartic acid (RY) has no agonistic activity in
any assay employed so far (16). RY is therefore a perfect high affinity
antagonist for human IL-4, as well as for IL-13, which also uses
IL-4R
for signal transduction (16, 17).
IL-4 antagonists may provide an effective way for the therapy of TH2-dominated diseases. The major obstacle for testing in vivo biological tolerance and therapeutic effects of IL-4 antagonists is the species specificity of IL-4. There is approximately 60% DNA sequence homology (18, 19) and no cross-reactivity between human and mouse IL-4 (20). Due to the lack of receptor binding, human IL-4 antagonists cannot be effective in mice. This has prompted us to develop an efficient antagonist for IL-4 in the mouse system.
BALB/c mice 6-8 weeks of age were purchased from Charles River/Wiga. The Spodoptera frugiperda insect cell line Sf9 was cultured in Insect Xpress medium (Serva, Heidelberg, Germany) without adjuvants at 27 °C in a 2-liter spinner flask aerated with 100% oxygen. Autographa californica nuclear polyhedrosis virus DNA (BaculoGoldTM DNA) was from Pharmingen (Hamburg, Germany).
Murine cells were cultured in RPMI 1640 containing 8% fetal calf serum, 100 units/ml penicillin and 100 µg/ml streptomycin, supplemented with 100 ng/ml IL-2 for CTLL-2 cells and with 5% culture supernatant of murine mIL-3-producing X63Ag8-653 BPV cells (21) for BA/F3 cells.
Production of IL-4 Wild Type and Double Mutant ProteinsRecombinant IL-4 proteins were produced in Sf9 cells
following Baculovirus infection. The pEP-B-splice mIL-4 plasmid
containing the cDNA for mouse IL-4 was kindly provided by Dr.
Werner Müller (University of Cologne, Germany) (22). A fragment
encoding the cDNA of mouse IL-4 was amplified by polymerase chain
reaction with the synthetic 5 primer 5
-CGCGGATCCCATATCCACGGA-3
and
the 3
primer 5
-GCCGGATCCTACGAGTAATCC-3
, comprising a
BamHI site and the first (for the 5
primer) and,
respectively, last (for the 3
primer) four codons of wild type IL-4.
The QY mutant gene was constructed using the 3
primer
5
-GCCGGATCCTACGAGTCATCCATATCCATGATGC-3
, resulting in substitution of
both glutamine 116 and tyrosine 119 by aspartic acid. The constructs
were sequenced and cloned into the Baculovirus transfer vector
pAc-GP67B (Pharmingen). The generation of recombinant viruses by
cotransfection of transfer plasmid and BaculoGoldTM DNA into insect
cells, and the selection of recombinant viruses by plaque assays were
performed as recommended by the manufacturer of the BaculoGoldTM
system. Cells were infected at a density of 2-4 × 106 cells/ml. The IL-4-containing supernatant was collected
72 h after infection. Escherichia coli-derived
recombinant IL-4 was from PeproTec (Rocky Hill, NJ).
Sf9 supernatant was extensively
dialyzed against 10 mM Tris/HCl, pH 8.0, followed by cation
exchange chromatography on a CM-Sepharose fast flow column (Pharmacia,
Freiburg, Germany) equilibrated with 10 mM Tris/HCl, pH
8.0, at 4 °C. Bound protein was eluted with a 0-1 M
NaCl gradient. The material was applied to a reversed phase high
pressure liquid chromatography column and eluted by a gradient of
acetonitrile from 30 to 50%. IL-4 and QY mutant were identified by
immunoblotting using rat anti-mouse IL-4 antibody (BVD4-1D11;
Pharmingen). The yield of purified recombinant protein was
approximately 1 mg/liter Sf9 supernatant. Freeze-dried protein was
stored at 20 °C and dissolved in H2O before use.
Protein was determined by the BCA method using bovine serum albumin as standard.
The recombinant
extracellular domain of mouse IL-4R (IL-4-BP) produced in Sf9 cells
was a kind gift of Dr. F.R. Seiler (Behringwerke AG, Marburg, Germany).
The protein (50 µg) was biotinylated by incubating with 6 nmol
NHS-LS-Biotin (Pierce, Oud Beijerland, Netherlands) in 250 µl of 0.1 M NaHCO3, pH 8.0, for 2 h at 0 °C. After purification by P6DG gelchromatography (Bio-Rad, München, Germany) in phosphate-buffered saline, the protein was frozen in
aliquots at
20 °C.
The biotinylated IL-4-BP was immobilized to a streptavidin-coated sensor chip CM5 (Pharmacia Biosensor AB, Uppsala, Sweden) at a density of 80-100 pg/mm2 in a BIAcoreTM 2000 system. The association and dissociation of IL-4 and QY in HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P-20) were analyzed at a flow rate of 50 µl/min and 25 °C. A set of sensograms was recorded at six different analytic concentrations. The data were evaluated using the BIA evaluation 2.1 software provided with the system as described (23). The rate constants evaluated from 15 independent measurements were used to calculate the mean values ± standard deviation.
Proliferation AssaysCytokine-induced proliferation of
cells was determined by [3H]TdR incorporation as
described (24). CTLL-2 cells were used at a density of 5 × 104/ml and BA/F3 cells at 5 × 105/ml.
T-cell-depleted splenic B-cells were prepared from BALB/c mice and
tested following standard procedures (25). Briefly, splenic cell
suspensions were incubated with rat anti-mouse Thy1.2 antibody (30H12;
Pharmingen) for 30 min, followed by incubation at 37 °C for 45 min
with Low-Tox®-H rabbit complement (Cedar Lane, Hornby, Ontario,
Canada). Viable and resting B-cells were separated on the 70-65%
interphase of a Percoll gradient (Pharmacia) and cultured at a density
of 106/ml with 5 µg/ml Salmonella typhimurium
LPS (Sigma, Deisenhofen, Germany) plus the indicated
concentrations of cytokines for 48 h. All measurements were
evaluated by means of the GraFit program (Erithacus Software) using the
equation y = a/(1+[X/I]expS) + back
(IC50 4 parameter logistic). The inhibitory constant
Ki was determined using the equation
Ki = ID50/(1 + [L]/ED50), where [L] is the concentration of ligand (IL-4), ID50 is
the concentration of QY required to decrease cellular response by half,
and ED50 is the IL-4 concentration required for
half-maximal cellular response.
Splenic B-cells (106/ml) were cultured in a 96-well plate with 5 µg/ml LPS and IL-4 or QY as indicated. Cells were centrifuged after 40 h, and supernatant was withdrawn. After blocking with 100 µl of phosphate-buffered saline, 3% bovine serum albumin for 1 h, cells were stained in opaque microtiter plates with 1 µg/well rat anti-mouse CD23 (B3B4; Pharmingen) at 4 °C for 1 h. Following incubation with goat anti-rat IgG-horseradish peroxidase conjugate for 45 min, detection was performed with an enhanced chemoluminescene based method (26) using a Microlumat LB 96-P luminometer (Berthold, Wildbad, Germany). ED50, ID50, and Ki were calculated as described above.
Tyrosine PhosphorylationProtein tyrosine phosphorylation was determined as described (17). Briefly, cells were incubated with cytokines and lysed, and proteins were precipitated with anti-phosphotyrosine antibody (4G10; Upstate Biotechnology Inc., Lake Placid, NY) or with anti-Stat6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Following SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes, anti-phosphotyrosine (RC20; Affinity, Nottingham, UK), anti-Stat6 (Santa Cruz) or anti-Jak3 antibody (Santa Cruz) were used for immunodetection.
Previous experiments have shown that the receptor binding affinity
of IL-4 is mainly determined by the high affinity interaction with
IL-4R. Therefore, the kinetics of interaction between mouse IL-4
proteins and the mouse IL-4R
chain were studied by means of a
BIAcoreTM 2000 system employing a recombinant extracellular domain,
IL-4-BP, immobilized to a biosensor matrix. The rate constants for the
dissociation of the complex between IL-4-BP and IL-4 or QY were
indistinguishable. The koff for both proteins
was about 2 × 10
3 s
1 (Fig.
1 and Table I). The association rate
constants were similar, but consistently a 50% lower on rate was found
for QY in comparison with IL-4. The dissociation equilibrium constant
Kd calculated as
koff/kon was about 400 pM for IL-4 and 800 pM for the QY variant.
|
The effects of the wild type and QY mutant protein on cell
proliferation were determined with highly purified splenic B-cells co-cultured with LPS, the T-cell line CTLL-2, and the pre-B-cell line
BA/F3. The biological activity of IL-4 produced by Sf9 cells was
identical to commercially available IL-4 produced by E. coli (Fig. 2A). IL-4 induced proliferation of
splenic B-cells, CTLL-2 cells, and BA/F3 cells (Fig. 2,
A-C). The ED50 values are summarized in Table
II. The QY mutant by itself had no detectable activity in proliferation assays (Fig. 2, A and B) but
inhibited IL-4-stimulated proliferation of all three cell types in a
dose-dependent fashion (Fig. 2, D-F). The
concentrations required for half-maximal inhibition of IL-4-induced
proliferation are given in Table II. As expected, in more sensitive
cells a higher dose of inhibitor was needed to block IL-4-induced
responses.
|
The low affinity IgE receptor, CD23, is an IL-4-inducible B-cell
differentiation marker. Half-maximal CD23 expression was induced by 19 pM IL-4 (Fig. 3A). Similiar to
the proliferation assays, there was no detectable activity of the QY
variant. QY prevented the IL-4-induced CD23 expression with
half-maximal inhibition reached at about 95-fold excess of the mutant
(Fig. 3B).
The signal transduction of cytokines involves the activation of various
tyrosine kinases and rapid phosphorylation of their substrates. IL-4
stimulates phosphorylation and activation of the transcription factor
Stat6 and the tyrosine kinase Jak3 (27). To determine whether the QY
mutant could inhibit IL-4 induced signaling, we have measured Stat6 and
Jak3 phosphorylation in BA/F3 cells. IL-4 at 1 nM
concentration induced tyrosine phosphorylation of both proteins,
whereas the same amount of the QY mutant did not stimulate
phosphorylation (Fig. 4). A 500-fold excess of the QY
variant completely inhibited the IL-4-induced response (Fig. 4).
Antagonistic properties of human IL-4 variants are caused by the deletion of a hydrophobic patch on the surface of the IL-4 molecule and by the introduction of an electrostatic mismatch (28). No local instability is introduced in the protein structure, because main chains structures of antagonistic mutants are identical compared with wild type (29).
No structural data on mouse IL-4 are available, but some mutation
studies have been reported. A deletion mutant of murine IL-4 lacking
residues 118-120 has impaired activity in a proliferation assay with
the T-cell line CT4R but retains high affinity binding to IL-4R
(30). Such a deletion, however, may alter the protein structure, and
inhibition of wild type protein was not tested. In the same study,
replacement of Gln116 with alanine had only minor effects
on receptor binding and biological activity (30). A murine IL-4 variant
with a single site mutation of tyrosine 119 (Y119D) has been mentioned
in the literature, but in one report no data were shown (31), and in
another one the same mutant induced agonistic effects similar to those
of wild type IL-4 in differentiation assays, like MHC II up-regulation on B-cells (32). This is to be expected for the Y119D mutant, because
murine and human
c interact in a structurally different way with
IL-4. Human IL-4 can productively interact both with human and murine
c, but human
c is most severely affected by mutations in
Tyr124 (13), and murine
c is most severely affected by
mutations in Arg121 (33). For this reason, and in
consideration of the partial agonist activities of human Y124D (13,
15), we decided to create the QY double mutant, as a murine analog to
the complete human IL-4 antagonist RY (16). As shown here, the QY
mutant was by itself inactive and completely antagonistic for wild type IL-4, in all assays performed.
Like IL-4, IL-13 can also direct IgE class switching in human B-cells (34, 35) but apparently not in the mouse, because IL-4 knockout mice are unable to produce IgE (36, 37). Anti-IL-4 antibodies or soluble IL-4 receptors may not be sufficient to treat for example type I hypersensitivity in humans, because neither would interfere with IL-13. Furthermore, both reagents can not only inhibit but also enhance effects of IL-4 by acting as carrier molecules that increase the lifetime of IL-4 in the serum (38). Antagonistic IL-4 mutants will inhibit both IL-4- and IL-13-mediated responses and cannot increase agonistic IL-4 effects. Because cytokine mutants are structurally nearly identical to the wild type protein, they should provide good immunological tolerance as well. The QY variant should allow to study such questions in vivo.