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Institut für Klinische Mikrobiologie, Immunologie, und Hygiene der Universität Erlangen-Nürnberg, Erlangen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/common 
chain (c chain)) and specifically inhibited the
proliferative effect of wild-type IL-4. In contrast, with IL-4.Y119D we
found induction of MHC class II and CD23 molecules on resting splenic B
cells as well as proliferation of B9 plasmocytoma cells. In addition,
IL-4.Y119D induced mRNA for soluble IL-4R, leading to the release of
soluble IL-4R protein by spleen cells. In macrophages, mutated IL-4 in
combination with IFN- induced TNF--dependent killing of
Leishmania major parasites such as wild-type IL-4. The
agonistic effects of IL-4.Y119D were observed on cells expressing the
IL-13R -chain, including an IL-13R -chain transfected T cell
line, but were absent in T cells that lack this molecule, indicating
that IL-4.Y119D conveys its activity via the type II IL-4R
(IL-4R/IL-13R). The described IL-4 mutant, therefore, represents
a new tool to use in dissecting different IL-4 functions that are
mediated by either type I or type II IL-4R
complexes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pleiotropic activities of IL-4 are mediated by high affinity
receptor(s) (dissociation constant (Kd) =
20–300 pM) that are expressed in relatively low numbers (100–5000) on
a variety of cell types (12). A majority of cells express the 140 kDa
IL-4R -chain in association with the common chain (c
chain)4 (13), which is
necessary, at least in T cells, for IL-4 signal transduction (14). The
trimolecular complex initiates the signal transduction by activation of
Janus kinase 1 (JAK1) and STAT6 via the IL-4R -chain and JAK3 via
the c chain (summarized in 15 , respectively. More recently,
IL-4 was demonstrated to induce functions on cells that express the
IL-4R -chain but lack the c chain (16, 17). These observations
indicate that there are at least two classes of IL-4R: type I IL-4R,
containing the IL-4R -chain and the c chain; and type II IL-4R,
consisting of the IL-4R -chain and IL-13-binding protein recently
identified and cloned by two research groups independently and
which associate with the IL-4R -chain (18, 19). Several
experimental findings support the hypothesis that these IL-13R
molecules are essential components of the type II IL-4R complex in the
sense of cytokine binding as well as signaling. First, a point-mutated
human IL-4 (IL-4.Y124D) displayed cross-competitive activity for both
human IL-4 and IL-13 (20). Second, blocking Abs against the human IL-4R
-chain also inhibited the effects of IL-13 (21). Third, IL-13 as
well as IL-4 was able to activate STAT6 in transfected CHO cells
expressing both IL-4R - and IL-13R -chains, while no activation
was observed in cells expressing one of these receptor molecules alone
(22).
Only two reports have been published so far concerning murine IL-4
mutants. Morrison and Leder (23) analyzed the receptor-binding domain
of mouse IL-4 using a solid-phase binding assay and in vitro
mutagenesis. They reported that the 16 amino-terminal residues and the
20 carboxyl-terminal residues are required for species-specific
receptor binding. Not surprisingly, IL-4 mutants no longer able to bind
to the receptor were also unable to induce T cell proliferation. More
recently, Grunewald et al. (24) expressed and characterized a double
mutant (IL-4.Q116D.Y119D) that completely antagonized all IL-4
functions tested, including the proliferation of T and B cells as well
as the up-regulation of CD23 on splenic B cells. This mutant,
therefore, resembles complete antagonists of human IL-4 previously
reported independently by two groups (20, 25). The generation of a
human IL-4 variant, by replacing tyrosine 124 with aspartic acid, led
to an antagonist that inhibited T and B cell proliferation (26) as well
as IL-4- or IL-13-induced IgG4 and IgE synthesis (27) and CD23
expression (26). Biochemical and structural evidence from
computer modeling of human IL-4R have shown that the site on human IL-4
defined by Y124 and R121 is required for the interaction with the human
c chain. In this study, we investigated the effect of a mutated form
of murine IL-4 with regard to wild-type IL-4 functions that have not
yet been studied, e.g., the expression of soluble IL-4R (sIL-4R) and
the enhancement of parasite killing by infected macrophages.
Additionally, we tested whether a differential effect on distinct IL-4
functions can be achieved by introducing a single point mutation into
mouse IL-4. The observations strongly suggest that the murine
IL-4.Y119D mutant is a type I IL-4R-specific antagonist.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female mice of the inbred strain BALB/c were obtained from Charles River Breeding Laboratories (Sulzfeld, Germany). IL-4-/- mice (Sv129 x C57BL/6) (8) as well as sex- and age-matched control animals were a generous gift from Dr. M. Kopf, Basel Institute for Immunology (Basel, Switzerland). TNFR55-/- mice (28) as well as sex- and age-matched control animals were kindly provided by Dr. K. Pfeffer (Munich, Germany).
Leishmania major promastigotes of the strain MHOM/IL/81/FEBNI were grown in vitro in blood agar cultures as previously described (29). Stationary phase promastigotes were washed in PBS and were added to the macrophage cultures, as indicated below.
Cytokines, Abs, and reagents
Recombinant murine IL-4 (LPS content <100 pg/µg protein) was
purchased from ICChemikalien (Ismaning, Germany) and recombinant murine
IFN- (LPS content <100 pg/µg protein) was from R&D Systems
(Wiesbaden, Germany). IL-2 was obtained from supernatants of
transfected X63Ag8-653 cells (30). Anti-murine IL-4 mAbs, BVD4-1D11,
and biotinylated BVD6-24G2 for the measurement of IL-4 in cell culture
supernatant by capture ELISA were obtained from PharMingen (Hamburg,
Germany), used as previously described (6) and as recommended by
the manufacturer. Purified rat anti-mouse CD23-biotin mAb and rat
anti-mouse I-Ad-FITC mAb were purchased from
PharMingen. Purified rat anti-mouse IL-4 mAb 11B11 was obtained
from Dianova (Hamburg, Germany), and Polymyxin B was from Sigma
(Deisenhofen, Germany). Purified recombinant murine and human sIL-4R
were kindly provided by Dr. Seiler (Behringwerke, Marburg, Germany).
Streptavidin-FITC was purchased from Boehringer Mannheim (Mannheim,
Germany).
Cloning and expression of murine IL-4.Y119D
The cDNA encoding murine IL-4.Y119D was generated by mismatch-PCR using murine IL-4 cDNA as template to introduce a point mutation leading to the tyrosine 119 to aspartic acid replacement. For expression in Escherichia coli, the sense primer 5'-CCGAATTCCATATCCACGGATGCGACAAAAAT-3' and the antisense primer 5'-GGCTCAGTACTACGAGTCATCCAT-3' were used. The resulting PCR fragment was purified by gel electrophoresis, digested with RsaI and SmaI, and cloned into His tag expression plasmid pQE30 (Qiagen, Hilden, Germany), which had been linearized with RsaI and SmaI. The resulting plasmid, pQE30-IL-4.Y119D, was transformed into the E. coli strain C600 (Stratagene, Heidelberg, Germany). To analyze the expression of IL-4.Y119D protein, transformed bacterial cells were induced with 2 mM isopropyl-ß-D-thiogalactoside for 1 to 5 h, and total bacterial proteins were analyzed on 15% SDS-PAGE. Optimized conditions of isopropyl-ß-D-thiogalactoside induction (3 h) were used for preparation of large amounts of rIL-4.Y119D. The bacterial cell pellets were mixed thoroughly in 2 volumes of homogenization buffer (50 mM Tris-HCl (pH 7.4)/10 mM MgCl2/0.2 M KCl/5% glycerol) and passed through a French press (SLM Aminco, Rochester, NY). The cell homogenates were centrifuged at 20.000 x g for 30 min at 4°C. The pellets were washed in 50 mM Tris-HCl (pH 7.4)/10 mM MgCl2 and treated with 6 M guanidine-HCl/0.1 M NaH2PO4/0.01 M Tris (pH 8.0) for 4 h at room temperature. The extracted IL-4.Y119D was affinity purified over Ni-NTA-resin (Qiagen) and eluted by reducing the pH, as recommended by the manufacturer. The denatured protein was refolded by slowly adding 9 volumes of 50 mM Tris-HCl (pH 7.4)/50 mM NaCl/0.005% Tween 20/2 mM reduced glutathione and 0.2 mM oxidized glutathione and incubated at room temperature for 4 h. The refolded protein was bound to a Hi-Trap affinity column (Pharmacia Biotech, Freiburg, Germany) coupled with anti-murine IL-4 mAb, 11B11, eluted with 100 mM glycine (pH 2), and dialyzed against PBS (pH 7.4).
For expression in a eukaryotic system, the following primers were used: sense primer, 5'-TCTCAACCCCCAGCTAGTTGTCAT-3'; and antisense primer, 5'-CCGAATTCCATATCCACGGATGCGACAAAAAT-3'. The PCR fragment was digested with BamHI and cloned into the BamHI linearized eukaryotic expression plasmid pCEP4 (Invitrogen, Leek, The Netherlands). The episomal pCEP4-IL-4.Y119D expression plasmid was transfected into the human embryonic kidney expression cell 293 EBNA (Invitrogen) by electroporation. Transfected cells selected by adding 300 µg/ml hygromycin B constitutively produced IL-4.Y119D. All cloned cDNAs were sequenced using the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer/Applied Biosystems, Warrington, U.K.) as recommended by the manufacturer.
Solid phase IL-4R binding assay
Purified human or murine rIL-4R was immobilized onto microtiter plates at a concentration of 5 µg/ml. After blocking nonspecific binding sites with FCS, serially diluted IL-4 or IL-4.Y119D was added to the wells, and the receptor-bound cytokine molecules were detected as described for the IL-4 ELISA applying the biotinylated mAb BVD6-24G2 (PharMingen).
Radioiodination of either wild-type IL-4 or IL-4.Y119D
Recombinant murine IL-4 or IL-4.Y119D was radiolabeled by the Iodogen (Pierce, Oud Bijerlad, The Netherlands) method as described by the manufacturer and by Lowenthal et al. (12). Briefly, 5 µg of purified cytokine was injected into glass reaction vessels coated with 0.5 µg of dried Iodogen before 2.5 µCi of 125I (Amersham, Braunschweig, Germany) was added. After 15 min at room temperature, an equal volume of phosphate buffer was added. The iodinated cytokines were purified by affinity chromatography as described above. BSA (0.1%) was added as a carrier protein to enhance 125I-IL-4 and 125I-IL-4.Y119D stability and to reduce loss caused by nonspecific adsorption to tubes.
Determination of 125I-IL-4- as well as 125I-IL-4.Y119D-binding affinity
The determination of the binding affinities of
125I-IL-4 and 125I-IL-4.Y119D to the IL-4R was
performed as described previously (12) using TF-1 cells stably
transfected with the murine IL-4R and expressing 
20,000 IL-4R
molecules per cell (31). Briefly, 3-fold serial dilutions of
125I-IL-4 and 125I-IL-4.Y119D were prepared in
100 µl (final volume) of complete Clicks RPMI medium in microfuge
tubes at 4°C. Then, 2 x 106 cells in 100 µl were
added to each microfuge tube and incubated at 4°C with gentle shaking
until binding equilibrium was reached (90 min). Cell-bound ligand was
separated from nonbound (free) by centrifugation of the cells through
an oil gradient. Nonspecific binding was measured in the presence of a
200-fold excess of unlabeled IL-4. Saturation binding curves and
Scatchard plots were calculated using GraphPad Prism software (San
Diego, CA).
Cell proliferation assays
D10.4G.1 (32), CTLL-2 (33), L1/1 (34), HT-2 (35), and B9 (36) were used for cell proliferation studies. The cells were propagated in complete medium (Clicks/RPMI medium (Life Technologies, Eppenstein, Germany) supplemented with 10% FCS (Biochrom, Berlin, Germany), 2 mM L-glutamine, 10 mM HEPES, 100 µg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3, and 5 x 10-5 M 2-ME) and stimulated in the presence or absence of various concentrations of IL-2, IL-4, and IL-4.Y119D, respectively, in 96-well flat-bottom microtiter plates (Nunc, Wiesbaden, Germany). D10.4G.1, CTLL-2, B9, and HT-2 cells were pulsed after 48 h of culture with [3H]thymidine (18.5 kBq/well; Amersham, Braunschweig, Germany) for 16 h and processed for beta counting. B9 and L1/1 cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 50 µg/well) for 4 h after 72 h of stimulation, stopped with 0.01N HCl/10% SDS, and colorimetrically analyzed at 550 nm.
Transfection of CTLL-2 cells with IL-13R expression constructs
cDNA prepared from B9 cells was used as template for PCR with
primers for the complete IL-13R -chain (sense primer,
5'-CCGGATCCGCGAGGGCCTGCATGGCGCGGCCA-3'; antisense primer,
5'-CCGAATTCCACTTCTCCCCATCAAGGAGCTGC-3'). The resulting cDNA
fragment was cloned into the
BamHI/EcoRI-linearized eukaryotic expression
vector pM5neo, which is based on the Moloney murine leukemia
virus and contains a neomycin resistance gene (kindly provided by W.
Ostertag, Hamburg, Germany). After confirmation of the nucleotide
sequence, 15 µg of purified receptor plasmid DNA was electroporated
into 1 x 107 CTLL-2 cells in 0.8 ml of medium at 900
µF and 260 V in an Easyject electroporation unit (Eurogentec,
Seraing, Belgium). Stably transfected cells were selected by
cultivation in the presence of 400 µg/ml G418 for 2 wk, then
subjected to single-cell cloning. IL-13R cell surface expression was
analyzed by flow cytometry, applying affinity-purified rabbit IgG
raised against the extracellular domain of the murine IL-13R -chain
expressed in E. coli.
B cell enrichment by magnetically activated cell sorting (MACS) and analysis by flow cytometry
B cells were purified from naive murine spleen cells by magnetic separation with a MACS column (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of the cells negatively selected with anti-CD4, -CD8, and -CD11b mAb-coupled microbeads (Miltenyi Biotech) was analyzed by flow cytometry leading to a 95 to 97% purity of B cells. Purified splenic B cells (1 x 106) were cultured in the presence or absence of different amounts of recombinant murine IL-4 and IL-4.Y119D for 16 h, respectively. The expression of CD23 and MHC class II was measured by flow cytometry.
In vitro stimulation of spleen cells
Spleen cells from IL-4-/- mouse were prepared as single-cell suspensions and cultured in vitro for 72 h at a density of 2 x 106/ml in complete medium in the presence or absence of various concentrations of IL-4 and IL-4.Y119D, respectively. After 72 h of culture, the release of the sIL-4R into the supernatant was determined by a specific sandwich ELISA as described previously (6).
RNA isolation and RT-PCR
After RNA extraction from murine lymphocytes with acidic
guanidinium thiocyanate (37), cDNA was synthesized for each time point
in 20-µl reactions containing 1 µg of total RNA, 50 mM Tris-HCl (pH
8.3), 5 mM MgCl2, 1 mM of each dNTP, 2.5 mM oligo(dT), 32 U
RNAguard, and 17 U AMV-RT (all from Pharmacia Biotech) at 42°C
for 90 min. The cDNA was amplified in a 40-µl reaction volume
containing 50 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 10 mM of
each dNTP, 1 U Taq polymerase (Pharmacia Biotech), and 100
nM primers during 35 cycles (1 min denaturation at 94°C, 1 min
annealing at 58–63°C, and 1 min extension at 72°C). Samples were
analyzed on 1.5% agarose gels containing 0.2 µg/ml ethidium bromide.
Primers used were as follows: spliced IL-4R sense primer,
5'-GCCCCAGTGGTAATGTGAAGCCCCT-3', and spliced IL-4R antisense primer,
5'-CTCACCACCGCAGCCCCCAAGGTCA-3' (amplified fragment of 373 bp); IL-13R
sense primer, 5'-ACAGAAGTTCAGCCACCTGTGACG-3', and IL-13R antisense
primer, 5'-CTAGGAGTTTTGCTCCTTACCTATACT-3' (amplified fragment of
941 bp); ß-actin sense primer, 5'-CACCCGCCACCAGTTCGCCA-3', and
ß-actin antisense primer, 5'-CAGGTCCCGGCCAGCCAGGT-3' (amplified
fragment of 574 bp); c chain sense primer,
5'-CCCAGAGAAAGAAGAGCAAGCACC-3', and c chain antisense primer,
5'-GGGGTCCTGGAGCTGGACAACAAA-3' (amplified fragment of 429 bp).
For quantification of the mRNA molecules coding for sIL-4R, a competitive PCR was used as described previously (38). Briefly, each cDNA was first amplified in the presence of diluted ß-actin control fragment (1:10 in a first approach followed by 1:2 dilution intervals for exact monitoring) of known concentration to determine the titer of control fragments needed to compete successfully with this cDNA. Then, relative concentrations of sIL-4R cDNA were determined in ß-actin-normalized cDNA samples using the same two-step protocol with a sIL-4R-specific control fragment. All reactions were repeated three times independently. The relative amounts of cDNA molecules were calculated and expressed on the basis of equivalent numbers of ß-actin cDNA molecules.
Stimulation of macrophages, infection with L. major, and assessment of intracellular parasites
Macrophage monolayers were prepared and tested for purity as described elsewhere (39). Briefly, thioglycolate-elicited peritoneal exudate cells (PEC) were seeded into Labtek tissue culture chamber slides (Nunc), and nonadherent cells were removed, after 4 h of incubation, by three intensive washings. Before infection, PEC were incubated for 4 h either in complete medium or in complete medium supplemented with cytokines as indicated in the text. The macrophages were then infected with L. major promastigotes (parasite:cell ratio, 8:1) in the respective media for 4 h. Thereafter, nonphagocytosed parasites were removed by two intensive washings, and the cultures were further incubated in the respective media for 72 to 96 h. Intracellular amastigotes were assessed by fluorescence microscopy, after staining with ethidium bromide (50 µg/ml) and acridine orange (5 µg/ml) in PBS, as described in detail elsewhere (39). The percentages of infected macrophages (infection rate) were determined. Differences between treated and control cultures were tested for statistical significance by Student’s t test for unpaired samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mutant murine IL-4 protein with an amino acid replacement of
tyrosine to aspartic acid near the carboxyl terminus at position 119
(Y119D) was cloned as described in Materials and Methods.
This protein was expressed in E. coli bacteria, as well as
in eukaryotic 293 EBNA cells, to analyze the influence of lack of
glycosylation, the degree of correct folding, and the influence of the
added His tag and to avoid LPS contamination of E.
coli-synthesized IL-4.Y119D. The two-step affinity purification of
the mutated IL-4 expressed in bacteria yielded an apparently homogenous
protein as judged by silver-stained SDS-PAGE, which could be detected
in Western blots with Abs specific for murine IL-4 as well as for the
His tag (Fig. 1
). The purified IL-4.Y119D
was folded correctly, since the neutralizing 11B11 mAb used for the
second affinity chromatography binds only to IL-4 possessing a correct
tertiary structure (data not shown). In addition, no differences with
regard to either the biologic effects of the IL-4 mutant or the
EC50 values measured were detected when IL-4.Y119D
expressed in 293 EBNA cells was used, suggesting that the glycosylation
of this protein is not critical for its biologic functions (data not
shown). In line with this finding, no difference between wild-type IL-4
produced in our laboratory (as glycosylated protein in 293 EBNA cells)
and the commercially obtained E. coli-expressed mouse IL-4
was detected in the different assays performed.
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First, the receptor-binding properties of the mutated IL-4 were
compared with those of the wild-type cytokine. In a solid phase binding
assay, applying either purified human or murine sIL-4R immobilized on
plastic surfaces, no differences of the resulting binding curves were
observed between IL-4 and IL-4.Y119D. Both proteins displayed specific
binding only to murine and not to human IL-4R, showing the species
specificity of the murine IL-4 forms (Fig. 2). To analyze ligand interactions with
IL-4R complexes, IL-4 and IL-4.Y119D were iodinated and tested for
binding to TF-1 cells expressing high numbers of IL-4R. Both IL-4 and
IL-4.Y119D displayed similar binding curves and
Kd values of 525 pM and 447 pM, respectively
(Fig. 3, A and B),
demonstrating that the affinity of IL-4 to its receptor is also not
influenced by the carboxyl-terminal amino acid substitution when the
IL-4R -chain is expressed on the surface of cells.
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-transfected T
cells
Although binding of IL-4.Y119D to the murine IL-4R -chain was
not affected as a result of this mutation, the IL-4 mutant was
unable to induce proliferation of several T cell lines, such as CTLL-2,
L1/1, HT-2 (Fig. 4), and D10.4G.1 cells
(data not shown); in contrast, these cell lines were highly responsive
to wild-type IL-4 (Fig. 4). To investigate whether IL-4.Y119D is able
to antagonize IL-4-induced proliferation, the T cell lines were
cultured with IL-4 or IL-2 in the presence of an excess of the IL-4
mutant. Indeed, IL-4.Y119D was a potent antagonist of wild-type IL-4,
leading to a complete inhibition of IL-4-induced T cell proliferation
in the presence of a sufficient (40-fold) molar excess (Fig. 5). The IL-2-induced proliferation of the
T cells was unaffected by IL-4.Y119D, thus excluding nonspecific or
toxic effects. To evaluate whether the presence of a type II
IL-4R complex leads to agonistic effects of IL-4.Y119D on T cells,
CTLL-2 cells were stably transfected with an expression vector encoding
the murine IL-13R -chain. Several isolated transfected clones
expressing the IL-13R not only displayed a proliferative response to
IL-13 but, more importantly, also to IL-4.Y119D. As shown in Table I, IL-13- and IL-4.Y119D-induced
proliferation reached comparable levels on IL-13R-expressing CTLL-2
cells, while no IL-13- and IL-4.Y119D-induced proliferation could be
measured on wild-type or control-transfected (pM5neo vector)
CTLL-2 cells. These experiments show that the reconstitution of a type
II IL-4R complex on these T cells confers responsiveness to the IL-4
mutant.
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Resting B cells were enriched from spleen cells of BALB/c mice
using MACS for the depletion of CD8+, CD4+, and
CD11b+ cells, yielding 95 to 97% pure B cell
populations. After 16 h of culture in the presence or absence of
IL-4 or IL-4.Y119D, respectively, the expression of CD23 and MHC class
II Ags was analyzed by flow cytometry. As depicted in Figure 6, A and B, both
surface molecules were up-regulated by wild-type IL-4 as well as
IL-4.Y119D in a dose-dependent manner, although the calculated
EC50 values indicated that IL-4.Y119D is 50-fold less
efficient than wild-type IL-4. Experiments with B cells from
IL-4-deficient mice showed similar results, excluding a significant
influence of endogenously produced IL-4 (data not shown).
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The murine plasmacytoma cell line B9 has been shown previously to
proliferate in response to IL-4 and IL-13 (40). Like wild-type IL-4,
the mutated form of IL-4 induced proliferation of B9 cells in a
dose-dependent manner (Fig. 7).
IL-4.Y119D-induced proliferation was independent of LPS contamination,
since it was also observed with IL-4.Y119D expressed in 293 EBNA cells,
which did not contain detectable amounts of endotoxin (LPS content
< 100 pg/µg protein). Furthermore, the addition of Polymyxin B into
the cultures of B9 cells stimulated with the E. coli-derived
protein did not influence IL-4.Y119D-induced proliferation (data not
shown). The calculated EC50 were 8.5-fold higher for the
IL-4.Y119D (EC50 = 170.6 ng/ml) as compared with wild-type
IL-4 (EC50 = 19.7 ng/ml), irrespective of whether MTT or
[3H]thymidine incorporation was measured. As expected,
due to the agonistic function of the IL-4 mutant, no inhibitory effect
on IL-13-induced proliferation of B9 cells was detected (data not
shown).
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-chain
The IL-13R -chain has been shown to participate in the
formation of functional type II IL-4R complexes, while the c chain
appeared to be indispensable for IL-4 effects mediated through the type
I IL-4R (14). Among the cell lines analyzed, only B9 cells expressed
mRNA for the IL-13R -chain, as detected with an IL-13R
-chain-specific primer pair, described in detail above, while mRNA
for the c chain was also detectable in the T cell lines (Fig. 8). The correlation between
IL-4.Y119D-induced cell proliferation and IL-13R -chain expression,
as well as the fact that IL-13R -chain transfected CTLL-2 cells
proliferated in response to IL-4.Y119D (see above), strongly argues for
a type II IL-4R-mediated effect of the IL-4 mutant and a type I
IL-4R-specific antagonism.
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An additional function of IL-4, which has been previously
demonstrated to be independent of the c chain, is the induction of
sIL-4R (38). IL-4 and IL-4.Y119D were added to spleen cell cultures
obtained from IL-4-deficient mice to avoid the complicating and
ill-defined effects of endogenously produced IL-4. As depicted in
Figure 9A, both forms of IL-4
stimulated the production of sIL-4R with similar kinetics. As already
known for IL-4 (38), the sIL-4R release induced by IL-4.Y119D was due
to the induction of the alternatively spliced form of IL-4R mRNA (Fig. 9A, inset).
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B). IL-4.Y119D stimulates the leishmanicidal effector function of murine macrophages
As reported previously (41), IL-4 (50 ng/ml) in combination
with IFN- (20 ng/ml) increases the killing activity of macrophages
to eliminate intracellular L. major amastigotes as long as
both cytokines are added simultaneously 4 h before infection (Fig. 10). This effect was not observed when
PEC from TNF R55-/- mice were used, confirming previous studies (42)
showing that the macrophage-stimulatory effect of IL-4 is mediated by
enhanced production of endogenous TNF-. Supernatants of transfected
293 EBNA cells were applied to analyze the effect of IL-4.Y119D on
L. major-infected macrophages (since the LPS-content of the
IL-4.Y119D produced in E. coli precludes experiments with
macrophages). These experiments clearly demonstrate that IL-4.Y119D,
like wild-type IL-4, is able to enhance the killing of intracellular
parasites in the presence of IFN- (Fig. 10). The percentage of
infected macrophages was reduced to a similar extent as with wild-type
IL-4, and this effect was not observed with macrophages obtained
from TNF R55-/- mice. The specificity was confirmed by the use of
supernatants of control-transfected 293 EBNA cells, which were
inactive, (Fig. 10) and by the inhibition of the IL-4.Y119D-mediated
leishmanicidal activity after adding 10 µg/ml neutralizing
anti-IL-4 mAb 11B11 (data not shown). The analysis of the
expression of the IL-13R -chain mRNA by RT-PCR showed that
this molecule is present in the adherent PEC (data not shown). Thus, it
is tempting to speculate that the effect of IL-4.Y119D on macrophages
might also be mediated independently of the c chain by type II
IL-4R.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The receptor-binding behavior of IL-4.Y119D was indistinguishable from
that of wild-type IL-4 in two types of assays. In addition to solid
phase binding assays, we iodinated both mutated and wild-type IL-4 to
directly measure the receptor binding on cells expressing high numbers
of functional IL-4R complexes. The observed Kd
values were not significantly different between IL-4 and its mutant,
which argues for an identical or very similar binding of IL-4.Y119D to
the heteromolecular IL-4R complexes, at least on the surface of these
cells. According to currently available models of the tertiary
structure of human IL-4, the critical tyrosine at position 119 of mouse
IL-4 is located on helix D of this 4-helix-bundle protein (43).
Cross-linking experiments using the human IL-4 double mutant
IL-4.R121D.S125D support the hypothesis that helix D of human IL-4
directly interacts with the c chain within the type I IL-4R (44).
Analogous to previous experiments with mutants of human IL-4 (20, 25, 26), we observed that the substitution of the most carboxyl-terminal
tyrosine abolished the T cell-stimulatory activity of mouse IL-4,
resulting in a competitive IL-4 antagonist on T cells. The
indispensability of the murine c chain for IL-4-induced growth of
mouse T cells has been defined previously by using mAbs to block this
receptor molecule (14). The observation that thymocytes from c
chain-deficient mice do not proliferate in response to IL-4 (45)
confirmed this conclusion. Together with our findings this suggests
that IL-4.Y119D antagonizes c chain-dependent functions mediated via
the type I IL-4R. In line with this hypothesis, IL-4 antagonism of the
IL-4 mutant is converted to agonistic functions on CTLL-2 cells
expressing a functional type II IL-4R after transfection with the
IL-13R -chain.
Interestingly, and in contrast to the results with T cells, IL-4.Y119D
induced the expression of CD23 and MHC class II on B cells as well as
the proliferation of B9 plasmocytoma cells. Three experimental findings
published by other groups indicate that the c chain is not
essentially involved in IL-4 effects on B cells. First, the induction
of MHC class II or CD23 molecules on murine B cells by IL-4 was
virtually unaffected by mAbs against the c chain (14). Second, B
cells deficient for the c chain derived from patients with X-SCID
were able to proliferate in response to IL-4 and IL-13 (16, 17). Third,
IL-4 was able to induce C
germline transcripts as well as IgE
isotype switching and, in addition, the dose-response curves to IL-4
for X-SCID and normal B cells were indistinguishable (46).
Interestingly, the human IL-4.Y124D mutant inhibited responses of
X-SCID B cells to IL-4 and IL-13 (17), clearly demonstrating that this
human IL-4 mutant is an IL-4 antagonist for functions mediated via
IL-4Rs that lack the c chain. Furthermore, indirect evidence
suggests that the up-regulation of CD23 on the surface of B cells is
also a c chain-independent function of IL-4. Oakes et al. (47)
reported that on human B cell lines from patients lacking JAK3, IL-4
induced CD23 expression, albeit less efficiently than on normal B
cells. Since it has been shown previously that JAK3 is activated
via the c chain (reviewed in 15 , these findings suggest that
CD23 expression might also occur in the absence of c chain
signaling. In purified splenic B cells, however, no signal for the
IL-13R could be detected by RT-PCR (data not shown). We thus
considered three possibilities by which IL-4.Y119D exerts its effects
on B cells in the absence of the c chain. First, the expression of
the IL-13R -chain, although of functional significance, might be too
low to be detected by RT-PCR, but this appears to be unlikely due to
the high sensitivity of the method. Second, in B cells as opposed to T
cells, IL-4 signaling might be initiated by homodimerization of IL-4R
-chains in the absence of additional receptor molecules. Third, B
cells might express an alternative IL-13R chain (IL-13R2), which has
recently been cloned from human cells. Since no sequence data on the
murine homologue have been published or deposited in gene data banks,
we are currently unable to distinguish experimentally between the two
latter possibilities.
Another cell type in which the c chain appears not to be essential
for the effects of IL-4 and IL-13 is the macrophage. Anderson et al.
(48) reported recently that IL-4 up-regulated MHC class II molecules
and inhibited nitric oxide production by macrophages from c
chain-deficient mice in response to LPS and IFN- to the same extent
as in wild-type macrophages. The c chain-deficient macrophages were
likewise fully responsive to IL-13, consistent with the model of a type
II IL-4R complex, which functions in the absence of the c chain. Of
special interest to the overall context of our study was the finding
that the mouse IL-4 double mutant IL-4.Q116D.Y119D completely
antagonized IL-4 and IL-13 responses on macrophages (48). Thus,
together with the three IL-4 functions previously reported to be
completely inhibited, e.g., the proliferation of T and B cells as well
as the up-regulation of CD23 on splenic B cells, the IL-4.Q116D.Y119D
double mutant is undoubtedly a complete type I and II IL-4R antagonist.
In contrast to this scenario, we demonstrate in this study that
IL-4.Y119D antagonizes IL-4 functions mediated through the c
chain-containing IL-4R type I complex but maintains c
chain-independent functions of IL-4, i.e., the stimulation of B cells
and macrophages, mediated via the type II IL-4R. However, the precise
molecular interactions of IL-4.Y119D with the different molecules of
the IL-4R complexes are still poorly defined, and especially the
analysis of the interaction of the mutant with the IL-13 -chain
awaits the development of reagents such as purified IL-13R
-chain or Abs directed against this molecule, which are not yet
available.
So far, we can only speculate on the reasons for the different dose-response relationships observed for the different effects of IL-4 and its mutant. The several IL-4 receptor chains and signal-transducing molecules such as STAT6, JAK1, JAK3, IRS-1, and IRS-2 might be either differentially expressed, involved in different cell types, and/or be of dissimilar importance for varied IL-4 effects such as up-regulation of cell surface molecules or the soluble IL-4R and cell proliferation.
To our knowledge, the only other example of a cytokine mutant distinguishing between two functional responses is human IL-5.E12K (49). This IL-5 mutant is a specific and potent antagonist for both IL-5-mediated proliferation of TF-1 cells and the adhesion of eosinophils. In contrast, IL-5.E12K is a full agonist in a human eosinophil survival assay, although with reduced potency compared with the wild-type protein. However, the exact molecular mechanisms leading to this split antagonism/agonism are not yet defined.
We think that the newly described murine IL-4.Y119D represents a
valuable tool for the dissection of different IL-4 functions in vitro
as well as in vivo. For example, there currently appears to be no other
way of distinguishing c chain-dependent from c chain-independent
IL-4 functions, since the complete inhibition of the c chain by Abs
or gene knock-out also definitively affects the function of other
cytokines that are dependent on this molecule, such as IL-2, IL-7,
IL-9, and IL-15. Thus, our future goal is to analyze the possible
therapeutic effects of IL-4.Y119D in the model of murine cutaneous
leishmaniasis, where certain IL-4 effects, such as the promotion of Th2
development, are disadvantageous for the host, while others, such as
the enhanced killing of parasites by IL-4-activated macrophages, are
desirable.
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Acknowledgments |
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Footnotes |
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2 Current address: Hoechst Marion Roussel, DG Rheumatology, P.O. Box 3540-65174, Wiesbaden, Germany.
3 Address correspondence and reprint requests to Dr. André Gessner, Institut für Klinische Mikrobiologie und Immunologie, Wasserturmstrasse 3, 91054 Erlangen, Germany. E-mail address:
4 Abbreviations used in this paper: c chain, common -chain; JAK1, Janus kinase 1; sIL-4R, soluble IL-4 receptor; MACS, magnetically activated cell sorting; PEC, peritoneal exudate cells; EC50, 50% effective concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 125I-IL-4, 125I-labeled IL-4.
Received for publication October 6, 1997. Accepted for publication June 1, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or
IL-4.Y119D (
), respectively. After 48 h,
[3H]thymidine was added, and 16 h later the cells
were harvested and radioactivity was measured in a beta counter. L1/1
cells were incubated with MTT, and the OD was measured at 550 nm.
X-174-RF DNA,
HincII digested); lanes 2, 8, and14, H2O controls; lanes 3, 9,
and 15, B9 cells; lanes 4, 10, and16, L1/1 Th2 cells; lanes 5, 11, and17, HT-2 cells; lanes 6, 12, and18, CTLL-2 cells; lanes 7, 13, and19, D10.4G.1 cells.
