A Murine Interleukin-4 Antagonistic Mutant Protein Completely Inhibits Interleukin-4-induced Cell Proliferation, Differentiation, and Signal Transduction*

(Received for publication, September 11, 1996)

Susanne M. Grunewald Dagger , Steffen Kunzmann , Bernd Schnarr , Juris Ezernieks , Walter Sebald and Albert Duschl §

From the Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Physiologische Chemie II, Am Hubland, D-97074 Würzburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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 alpha  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.


INTRODUCTION

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 epsilon  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 alpha  chain (IL-4Ralpha ) binds IL-4 with high affinity (6-8). The second subunit is the 64-kDa common gamma  receptor subunit (gamma 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-4Ralpha (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-4Ralpha binding but can severely impair cellular responses, because they result in loss of interaction with gamma 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-4Ralpha 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.


MATERIALS AND METHODS

Animals, Cells, and Viruses

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 Proteins

Recombinant 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).

Protein Purification

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.

In Vitro Receptor Binding Assay

The recombinant extracellular domain of mouse IL-4Ralpha (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 Assays

Cytokine-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.

Determination of CD23 Expression

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 Phosphorylation

Protein 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.


RESULTS AND DISCUSSION

Previous experiments have shown that the receptor binding affinity of IL-4 is mainly determined by the high affinity interaction with IL-4Ralpha . Therefore, the kinetics of interaction between mouse IL-4 proteins and the mouse IL-4Ralpha 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.


Fig. 1. Sensograms showing the binding and release of IL-4 and QY variant to and from immobilized IL-4-BP. 5 nM IL-4 or 10 nM QY were perfused over IL-4-BP, immobilized at a biosensor matrix (0-300 s). Dissociation commenced after perfusion with buffer alone (>300 s). Results are given as resonance units (RU). The kinetics of binding were comparable for IL-4 and QY, except for a 50% lower on rate for QY, which was compensated in the experiment shown by using a two times higher concentration of QY compared with IL-4.
[View Larger Version of this Image (11K GIF file)]


Table I.

Dissociation (koff) and association (kon) rate constants of IL-4 and the QY variant with IL-4-BP

Association and dissociation rates for the binding of mouse IL-4 and QY to IL-4-BP, as determined by biosensor experiments. The data are mean values (n = 15) ± standard deviation. The results from two independent experiments (Exp.) are shown for each protein.
kon koff Kd

m-1 s-1 s-1 pM
IL-4 Exp. 1 4.6  ± 0.69 × 106 1.8  ± 0.36 × 10-3 390
Exp. 2 5.1  ± 0.77 × 106 1.9  ± 0.48 × 10-3 340
QY Exp. 1 2.2  ± 0.37 × 106 1.8  ± 0.54 × 10-3 820
Exp. 2 2.3  ± 0.60 × 106 1.9  ± 0.57 × 10-3 830

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.


Fig. 2. Effects of QY mutant on IL-4-induced cell proliferation. Cells were stimulated as indicated, and DNA synthesis was measured after 24 h. A-C, proliferation in response to increasing doses of IL-4 or QY mutant. D-F, competitive inhibition of IL-4-induced proliferation by the QY mutant. A and D, LPS-activated splenic B-cells; B and E, pre-B-cell line BAF/3; C and F, T-cell line CTLL-2. bullet , IL-4 (Sf9); black-triangle, IL-4 (E. coli); ×, QY; open circle , QY + IL-4; square , medium control.
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Table II.

Effective and inhibitory doses of IL-4 and QY mutant for proliferation in different cell types

IL-4 concentration required for half-maximal proliferation (ED50) and QY concentration required for half-maximal inhibition (ID50). The data shown are mean values ± standard deviation.
Cell type Mean ED50 Mean ID50 Mean Ki

pM nM pM
BA/F3 67  ± 60 (n = 7) 3  ± 0.7 (n = 6) 190  ± 150 (n = 6)
Splenic B-cell 16  ± 10 (n = 3) 37  ± 0.9 (n = 3) 600  ± 380 (n = 3)
CTLL-2 9  ± 7.4 (n = 7) 61  ± 53 (n = 2) 520  ± 300 (n = 2)

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).


Fig. 3. Effects of QY mutant on IL-4-induced CD23 expression of splenic B-cells. Cells were stimulated with LPS and cytokines in the indicated concentrations and CD23 expression was determined after 72 h. A, induction of CD23 by increasing concentrations of IL-4. B, competitive inhibition of IL-4-induced CD23 expression by increasing doses of QY mutant. bullet , IL-4; open circle , QY + IL-4.
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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).


Fig. 4. IL-4-induced tyrosine phosphorylation. Factor-deprived BA/F3 cells were stimulated for 10 min with cytokines in the indicated concentrations. IL-4-induced tyrosine phosphorylation of Jak3 and Stat6 was shown by immunoprecipitation and Western blotting.
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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-4Ralpha (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 gamma c interact in a structurally different way with IL-4. Human IL-4 can productively interact both with human and murine gamma c, but human gamma c is most severely affected by mutations in Tyr124 (13), and murine gamma 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.


FOOTNOTES

*   This work was supported by the Deutsche Forschungsgemeinschaft (Du 220/2-1), by the Jubiläumsstiftung der Industrie und Handelskammer Würzburg-Schweinfurt, and by the Senator Kurt und Inge Schuster Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Recipient of a scholarship from the Deutsche Forschungsgemeinschaft.
§   To whom correspondence should be addressed. Tel.: 49-931-888-4117; Fax: +9-931-888-4113; E-mail: duschl{at}biozentrum.uni-wuerzburg.de.
1    The abbreviations used are: IL, interleukin; IL-4Ralpha , 140 kDa alpha  chain of the IL-4 receptor; IL-4-BP, recombinant extracellular domain of IL-4Ralpha ; gamma c, common subunit of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15; LPS, lipopolysaccharide.

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