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* This investigation received financial assistance from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases Grant 970551.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. ** Supported by Swiss National Science Foundation Grant 32-63550.00.
The eggs of the parasitic trematodeSchistosoma mansoni are powerful inducers of a T helper type 2 (Th2) immune response and immunoglobulin E (IgE) production.S. mansoni egg extract (SmEA) stimulates human basophils to rapidly release large amounts of interleukin (IL)-4, the key promoter of a Th2 response. Here we show purification and sequence of the IL-4-inducing principle of S. mansoni eggs (IPSE). Stimulation studies with human basophils using SmEA fractions and natural and recombinant IPSE as well as neutralization and immunodepletion studies using antibodies to recombinant IPSE demonstrate that IPSE is the bioactive principle in SmEA leading to activation of basophils and to expression of IL-4 and IL-13. Regarding the mechanism of action, blot analysis showed that IPSE is an IgE-binding factor, suggesting that it becomes effective via cross-linking receptor-bound IgE on basophils. Immunohistology revealed that IPSE is enriched in and secreted from the subshell area of the schistosome egg. We conclude from these data that IPSE may be an important parasite-derived component for skewing the immune response toward Th2.
T helper type 2
expressed sequence tag
high affinity IgE receptor
interleukin-4-inducing principle from S. mansoni eggs
saline-solubleS. mansoni egg antigen extract
S. mansoni egg cDNA library
regulated on activation normal T cell expressed and secreted
Infection with the parasitic trematode Schistosoma mansoni leads to a pronounced Th21 response and to elevated IgE production both in humans and in experimental animals. The definition of parasite-derived products capable of skewing the immune response toward Th2 would not only enhance our understanding of the defense mechanisms involved in helminth infections but may also lead to new insights into the pathogenesis of immediate-type hypersensitivity diseases such as asthma. However, in contrast to our increasing understanding of how pathogen-derived products can initiate Th1-type immune responses, there is so far little detailed knowledge about the nature of the parasite-derived molecule(s) and the underlying mechanisms that trigger and/or amplify a Th2-type reaction. In S. mansoni infection, a critical role in inducing a polarized Th2 response is played by the egg stage of the parasite (
) into naive animals. By contrast, the initial larval (schistosomula) and adult worm stages rather induce a response skewed to Th1.
It is now firmly established, both in vivo and in vitro, that the cytokine profile present during an immune reaction is an important element in directing the response to Th1 or Th2 and that IL-4 is the key cytokine responsible for biasing the immune reaction toward a Th2 phenotype (
). In the human system, basophils are a prominent source of IL-4 and IL-13; these cells secrete large amounts of IL-4 and IL-13 in response to IgE-receptor cross-linking or activation by a combination of IL-3 and C5a (
). Indeed, human basophils can be viewed as “innate Th2-type” effector cells, since IL-4 and IL-13 are expressed in a very restricted manner without production of any of the cytokines involved in Th1-type immune responses. We therefore wondered whether saline-soluble S. mansoni egg antigen extract (SmEA) can directly trigger human blood basophils to release IL-4, thus providing a potential mechanism for biasing the immune response to Th2 during the egg stage of S. mansoni infestation. Indeed, our previous studies showed that basophils from healthy nonsensitized donors from Northern Europe rapidly degranulate and release histamine, sulfidoleukotrienes, and considerable amounts of IL-4 as well as IL-13 in response to exposure to SmEA (
SmEA thus obviously contains a basophil-degranulating IL-4-inducing principle, prompting us to further characterize this activity. Subsequent studies revealed that the active principle is an excretory/secretory glycoprotein interacting with IgE on basophils in an antigen-nonspecific way (
Here we present the isolation, sequencing, and cloning of the bioactive product, which was named IL-4-inducing principle from S. mansoni eggs (IPSE). Finally, as unambiguous proof of the identity of the cloned S. mansoni product, we demonstrate that the recombinant protein has full biological activity.
S. mansoni eggs were purified as previously described (
) and used at a mean purity of 99%. Purified basophils were incubated in round-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) at 37 °C in humidified air containing 6% CO2 at 106 basophils/ml in a final volume of 100 μl of culture medium (Iscove's modified Dulbecco's medium supplemented with 100 units/ml penicillin G, 50 μg/ml transferrin, 5 μg/ml insulin, 100 μg/ml streptomycin, 10% fetal calf serum). Since IL-3 enhances Fcε RI-mediated (
) IL-4 production of basophils, all wells were supplemented with IL-3 except the wells to which ionomycin was added. The cells were incubated with various stimuli at the concentrations indicated. If not stated otherwise, the supernatants were collected after 18 h and stored at −20 °C until further investigation. Degranulation of basophils was assessed via cytospin preparation and May- Gruenwald staining.
Cytokine and Histamine Assays
IL-4 and IL-13 levels in culture supernatants were determined using two-site sandwich enzyme-linked immunosorbent assays (Eli-Pair; Diaclone, Besancon, France) essentially according to the manufacturer's protocol. The sensitivities for IL-4 and IL-13 detection were 0.55 and 1.56 pg/ml, respectively. Histamine was determined using the methyl-histamine radioimmune assay (Amersham Biosciences) according to the manufacturer's recommendations.
SDS-PAGE and Blotting
Proteins were separated by SDS-PAGE (12% T, 4% C) under reducing and nonreducing conditions according to Laemmli (
). Free binding sites were blocked by incubating the membrane with 0.05% (v/v) Tween 20 in 0.1 mTris-buffered saline, pH 7.4. The membranes were then incubated with alkaline phosphatase-labeled Aleuria aurantia agglutinin (AAA; 1:5000; Vector Laboratories, Burlingame, CA) or with antibodies (at the concentrations indicated) in 0.1 m Tris-buffered saline, pH 7.4, containing 0.05% (v/v) Tween 20. Lectin and antibody binding was visualized by a substrate/chromogen mixture of 0.033% (w/v) nitro blue tetrazolium and 0.017% (w/v) 5-bromo-4-chloro-3-indolyl phosphate (Serva, Heidelberg, Germany) in 0.1 m Tris-buffered saline, pH 9.5 (
IPSE was purified by cation exchange and affinity chromatography. First, 2 mg of SmEA in 20 mm potassium phosphate buffer, pH 5.0, were bound to a HiTrap SP-Sepharose column (1 ml; Amersham Biosciences). Elution of bound proteins was carried out using a linear salt gradient from 0 to 1 m KCl. Effluent and eluate fractions were analyzed for their IL-4-inducing effect (applied volume: 5 μl/106 basophils) and (after speedvac concentration) for their protein banding pattern. The latter was determined by SDS-PAGE followed by silver staining. Fractions containing IPSE were pooled and concentrated, and for affinity chromatography, the buffer was exchanged to 50 mm sodium phosphate buffer, pH 7.0, by using Centricon Plus-20 centrifugal filters (Millipore Corp., Bedford, MA).
For affinity chromatography, AAA (2 mg; Vector Laboratories) was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap columns (Amersham Biosciences) according to the manufacturer's instructions. After application of pooled IPSE-containing fractions and washing, bound material was eluted with 0.1 ml-fucose in 50 mm sodium phosphate buffer, pH 7.0. Fractions containing IPSE were pooled, concentrated by Centricon Plus-20 centrifugal filters, and stored at −80 °C.
N-terminal Sequencing of Purified IPSE
N-terminal sequencing of the first 20 aa of the purified IPSE was performed on an Procise 492 Sequencer (Applied Biosystems) at Chromatec Ltd. (Greifswald, Germany).
S. mansoni Egg cDNA Libraries (SmE-libraries)
mRNA was isolated from 1 × 106S. mansoni eggs using the μMACS mRNA isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. 3 μg of mRNA were obtained and used for construction of a cDNA library in λ ZAP II by means of the Lambda ZAP II XR library construction kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. The resulting cDNA library of S. mansoni eggs had 70% inserts and a titer of 6 × 109 plaque-forming units/ml.
The other SmE-library was constructed as previously described (
Screening of SmE-libraries Using a Dig-labeled Oligonucleotide
For screening of the cDNA libraries, a sequence-specific 30-mer oligonucleotide (for sequence, see Fig. 3) was labeled with Dig-ddUTP using the Dig oligonucleotide 3′-end labeling kit (Roche Diagnostics). The labeled oligonucleotide was detected via an AP-conjugated anti-Dig antibody (1:5000; Roche Diagnostics). Plaque lifts and hybridization were performed as described by Sambrook and Russell (
). Hybridization was carried out overnight at 55 °C in 5× SSC, 1% (w/v) blocking reagent (Roche Diagnostics), 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS. Prior to detection of bound oligonucleotide, filters were washed twice for 5 min at room temperature with 2× SSC, 0.1% (w/v) SDS and twice for 15 min at 55 °C with 0.2× SSC, 0.1% (w/v) SDS. Positive phage clones were isolated, and the pBluescript SK plasmids containing the inserts were obtained by in vivo excision using the helper phage Exassist (Stratagene). Automated sequencing of double-stranded plasmid DNA was performed by cycle sequencing using the Big Dye Terminator Cycle Sequencing ready Reaction Kit on an ABI 377 DNA sequencer (PerkinElmer Life Sciences).
Expression and Purification of Recombinant IPSE in Escherichia coli
Recombinant IPSE was expressed as a His tag fusion protein (His-IPSE) in E. coli. cDNA coding for IPSE was subcloned into the pProEX HTb expression vector (Invitrogen) coding for 6 N-terminally located histidine residues and a cleavage site for TEV protease. For this purpose, PCR was performed with IPSE-specific primers containing an AT clamp and the following restriction sites (underlined): EheI for the sense primer (5′-ATATATGGCGCCGATTCATGCAAATATTGTC-3′) andHindIII for the antisense primer (5′-ATATATAAGCTTTCATCAGTTCATATGC-3′), respectively. Conditions for PCR were as follows: denaturation for 30 s at 95 °C, annealing for 30 s at 44 °C, and extension for 1 min at 72 °C (30 cycles). Subcloning and confirmation of DNA sequences followed standard procedures (
). E. coli cultures containing the recombinant plasmid were grown at 37 °C to anA600 of 0.8, and then expression of His-IPSE was induced by 0.6 mm isopropyl thiogalactoside. After incubation for an additional 4 h, cells were harvested by centrifugation and resuspended in 30 ml 50 mm Tris/HCl, pH 8.0, 0.8 mm EDTA, 200 mm NaCl, 25% sucrose. His-IPSE was recovered as insoluble inclusion bodies after breaking the cells by passaging them twice through a French press (at a gauge reading of 1000 p.s.i.) and subsequent centrifugation at 7000 × g. The insoluble fraction was washed once with 20 mm Tris/HCl, pH 8.0, 0.5% deoxycholate and once with 20 mm Tris/HCl, pH 8.0, and subsequently solubilized in 8m urea, 500 mm NaCl, 50 mm sodium phosphate, pH 7.5. His-IPSE was purified by nickel-nitrilotriacetic acid metal affinity chromatography (Qiagen, Hilden, Germany) under denaturing conditions according to the manufacturer's protocol. Using the Hampton Research FoldIT Screen (Hampton Research, Laguna Niguel, CA), optimal buffer conditions for refolding of the denatured His-IPSE were obtained. Briefly, purified His-IPSE was refolded by dialysis at a concentration of 0.1 mg/ml against FoldIT buffer 3 (modified to pH 10.5) and subsequently against a physiological buffer such as phosphate-buffered saline (PBS).
Preparation of Monoclonal Antibodies and Murine Antiserum to IPSE
Monoclonal antibodies and murine antiserum to His-IPSE were raised by Labsoft Diagnostics AG (Halle, Germany). To obtain monoclonal antibodies, aliquots containing 28 μg of His-IPSE emulsified in 100 μl of saline (154 mm) and 300 μl of complete Freund's adjuvant were intraperitoneally administered to 10-week-old female BALB/c mice. The intraperitoneal immunization was repeated after 9 weeks (28 μg of antigen with incomplete adjuvant). The third (after further 2 weeks) and the fourth injection (3 days thereafter) of IPSE (each 28 μg) were administered in saline (murine antiserum to IPSE was raised by a similar immunization protocol). One day after the last booster injection, the animals were killed and blood was taken for antiserum preparation. The fusion was performed according to a standard procedure using the mouse nonsecretory cell line P3X63Ag8.653 as fusion partner.
Immunohistological Detection of IPSE
Liver sections (formalin-fixed, paraffin-embedded material) of mice infected with 150 S. mansoni cercariae 7 weeks previously were incubated overnight with the monoclonal anti-IPSE antibody 74 1G2 (undiluted culture supernatant) or (as negative controls) with murine IgG (100 μg/ml in PBS; Dianova, Hamburg, Germany) or PBS only. Subsequently, the sections were incubated for 1 h with AP-conjugated F(ab′)2 goat anti-mouse IgG antibodies (1:100; Dianova), followed by staining with Neufuchsin (Dako, Hamburg, Germany) and hematoxylin (Merck). All steps were carried out according to routine protocols.
Computer Analysis and Nucleotide Sequence Accession Number
Sequence data were analyzed with DNASIS software (Amersham Biosciences). The GenBankTM data base (NCBI) was searched for homologous sequences using the nucleotide and protein BLAST programs. Secondary structure predictions were performed online using the “Protein Predict server” available on the World Wide Web at maple.bioc.columbia.edu/predictprotein. The nucleotide sequence of IPSE was submitted to the GenBankTM data base and has been assigned GenBankTM accession number AY028436.
Here we describe the purification, N-terminal sequence analysis, cloning, and recombinant expression of IPSE, a glycoprotein secreted from S. mansoni eggs. IPSE triggers basophils from naive human donors to rapidly degranulate, release mediators, and express IL-4 and IL-13, the key cytokines controlling a Th2-type immune response and IgE synthesis.
The protocol for purifying IPSE was a combination of cation exchange chromatography and affinity chromatography with the lectin AAA. The purified material had a high specific activity and consisted of two bands of about 40 kDa as assessed by nonreducing SDS-PAGE. The purity of the natural bioactive material was further demonstrated by microsequencing a preparation containing the two bands, which revealed one unequivocal N-terminal aa sequence, suggesting that both represent posttranslational variants of the same protein. This conclusion was supported by the finding that all clones (n = 12) obtained from the two libraries were 100% identical.
For probing the SmE-libraries, a nondegenerate 30-mer oligonucleotide was used, corresponding to a region of 10 aa that was identical between the N-terminal sequence of purified IPSE and the homologous S. mansoni EST retrieved from the data base. Thus, if present, the cDNA corresponding to the EST should also have been recovered. However, the fact that from two different SmE-libraries 12 identical clones corresponding to IPSE were isolated suggests that the respective sequence represents a dominant form of this molecule, whereas another highly homologous S. mansoni gene either does not exist or is expressed in the egg stage only at a very low level.
The mature protein consists of 114 aa, without relevant sequence homologies with known proteins. Secondary structure predictions (PHD/PROF on the World Wide Web at maple.bioc.columbia.edu/predictprotein) indicate that the IPSE monomer forms a compact globular structure composed mainly of β-sheets, including a crystallin β/γ “Greek key” motif. Thus, IPSE may have some structural similarity with the γ-crystallin-like superfamily, and indeed an assignment to this superfamily was also obtained (E value: 4.4e−05) by using the Superfamily HMM Library and Genome Assignments server (available on the World Wide Web at supfam.org/SUPERFAMILY). As assessed by SDS-PAGE analysis performed under reducing and nonreducing conditions, recombinant IPSE spontaneously formed disulfide-linked homodimers, consistent with the presence of an uneven number of seven cysteines. Correspondingly, when natural IPSE was subjected to SDS-PAGE under reducing conditions, its apparent molecular mass decreased from 40 to 20 kDa. Given a calculated molecular mass of 26.4 kDa for recombinant homodimeric IPSE, natural IPSE appears to be a homodimeric glycoprotein with a glycan content of about 30%.
Clear evidence that the recombinant protein corresponds to IPSE came from functional studies. Like natural IPSE, the recombinant analogue triggered degranulation of basophils and caused release of histamine, IL-4, and IL-13. The high specific activity of His-IPSE (maximum at 0.75 nm) was in the same range as that of the natural protein. Moreover, the fusion protein formed homodimers just like natural IPSE. Cell activation experiments with whole SmEA, pooled SmEA fractions, and recombinant IPSE, respectively, strongly suggested that basophil activation by SmEA was due to IPSE and not to other components of SmEA. In line with these findings, agglutination of basophils correlated with the presence of IPSE and not the other components. Moreover, antibodies raised against His-IPSE were reactive with natural IPSE (Western blot) and functionally neutralized as well as immunodepleted natural IPSE from SmEA.
Although IPSE seemed to be a completely novel protein, the similarity of its appearance in cation exchange fractions and its apparent molecular weights under reducing and nonreducing conditions as well as its glycosylation suggested that IPSE might be identical with antigen α1 found in S. mansoni eggs (
). This glycoprotein is a candidate antigen for serodiagnosis of S. mansoni infection but had thus far not been further characterized. Indeed, Western blotting of antigen α1 and IPSE, respectively, with anti-α1 and anti-IPSE antibodies as well as N-terminal sequencing of antigen α1 clearly indicates both molecules to be identical.
) and are also required for SmEA-stimulated Th2 induction. The latter was demonstrated in a murine model of intranasal sensitization with deglycosylated (periodate-treated) SmEA that, compared with the native material, failed to induce IL-4, IL-5, IL-10, and SmEA-specific IgE production (
). Moreover, lacto-N-fucopentaose III, a predominant glycan component of SmEA that contains Lewisx (Lex), has been found to function as a Th2-inducing adjuvant in mice when covalently linked to human serum albumin (
), it is possible that natural IPSE contains lacto-N-fucopentaose III. However, for the functional activity of IPSE being investigated here, carbohydrates are not necessary, since unglycosylated recombinant IPSE has a powerful IL-4-inducing effect. Moreover, lacto-N-fucopentaose III coupled to human serum albumin (Biocarb Chemicals, Lund, Sweden) did not activate human basophils when added over a wide concentration range (0.03–30 μg/ml using 0.3 log increments; data not shown). Of course, our findings do not exclude the possibility that carbohydrates can contribute to a Th2 bias by mechanisms involving cell types other than basophils.
Regarding the mechanism by which IPSE activates basophils, earlier IgE stripping and resensitization experiments had revealed that IgE has to be present on the cells for IPSE to exert its effect (
), suggesting that this schistosome-derived molecule acts via cross-linking of Fcε RI-bound IgE. This assumption is supported by our blotting and Biacore experiments demonstrating dose-dependent binding of His-IPSE to solid-phase IgE andvice versa. Taken together, IPSE is an IgE-binding factor that activates basophils presumably via cross-linking receptor-bound IgE.
The type of interaction between IPSE and IgE is not yet known. Basically, it could be an antigen-specific, lectin-like, or B cell superantigen-like interaction. Antigen-specific binding is highly unlikely, since the basophils were from nonsensitized donors, and, in the case of the recombinant molecule, potentially cross-reactive carbohydrates were not involved. The possibility that IPSE acts as a lectin cannot be excluded, but its sequence does not show any similarity with known animal or plant lectins. Furthermore, although several lectins can activate basophils (
), the concentrations required are in the μg/ml range. In contrast, the possibility that IPSE is a B cell superantigen requires consideration; B cell superantigens, such as HIV-1 gp120, protein Fv, and protein L (
),2 can bind to IgE and, as multivalent molecules, can activate basophils, resulting in the release of IL-4 and IL-13. Furthermore, release of IPSE-induced IL-4 can be inhibited in the presence of increasing concentrations of human polyclonal IgG. Polyclonal IgG contains various VH segment family members, and some of them may compete with corresponding VH segments of IgE for binding to IPSE. Experiments addressing these points are currently under way.
The in vivo effects of IPSE are not known yet. The pronounced IL-4 and IL-13 production triggered by this factor in vitro strongly suggests that IPSE might be involved in initiating and/or amplifying a Th2 response. Basophils, while accounting for only 1% of human peripheral blood leukocytes (
). Basophils are mobile blood cells and can readily reach sites of interaction with pathogens; they may thus be attracted to sites of schistosome egg deposition. Morphologically intact granulated basophils are rarely observed in schistosome egg granulomas, but in view of the powerful activating effect of IPSE, it is quite likely that basophils in this environment are degranulated (“ghost cells”) and thus are not easily recognized by conventional histochemical staining. Moreover, basophils share several chemokine receptors with eosinophils such as CCR2 and CCR3 (
), which are bound by the chemokines MCP-1, MCP-2, MCP-3, and MCP-4 and the chemokines MCP-3, MCP-4, RANTES, eotaxin I, and eotaxin II, respectively, suggesting that they could be important participants in the circumoval immune response, which is dominated by eosinophils for several weeks. Since IPSE is secreted by schistosome eggs, as concluded both from its presence in egg culture supernatants and from the immunohistological finding of IPSE on and in circumoval inflammatory cells, this factor will obviously interact also with basophils attracted to the schistosome egg granuloma.
In conclusion, IPSE is a parasitic glycoprotein with a novel and potent activating effect on human basophils. Because it can rapidly trigger basophils from nonsensitized human donors to release considerable amounts of IL-4, IPSE is a good molecular candidate for biasing the immune response toward the Th2 phenotype in the course of schistosome infection.
We are grateful to A. Ruppel, M. Kastner, W. Kunz, M.-Q. Klinkert, G. Dörfler, U. Zähringer, T. Goldmann, and L. Rink for advice and/or practical help and to W. Martens and S. Adrian for excellent technical assistance.