Molecular Characterization of an Interleukin-4-inducing Factor from Schistosoma mansoni Eggs

Abstract

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. 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 interleukin amino acid(s) A. aurantiaagglutinin expressed sequence tag high affinity IgE receptor interleukin-4-inducing principle from S. mansoni eggs phosphate-buffered saline saline-solubleS. mansoni egg antigen extract S. mansoni egg cDNA library digoxigenin alkaline phosphatase 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 (1Pearce E.J. Caspar P. Grzych J.-M. Lewis F.A. Sher A. J. Exp. Med. 1991; 173: 159-166Crossref PubMed Scopus (684) Google Scholar), since a Th2 response and IgE production are only observed after egg deposition or following injection of schistosome eggs (2Vella A.T. Pearce E.J. J. Immunol. 1992; 148: 2283-2290PubMed Google Scholar) or extracts thereof (3Okano M. Nishizaki K. Abe M. Wang M.M. Yoshino T. Satoskar A.R. Masuda Y. Harn Jr., D.A. Allergy. 1999; 54: 593-601Crossref PubMed Scopus (55) Google Scholar) 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 (4Paul W.E. Seder R.A. Cell. 1994; 76: 241-251Abstract Full Text PDF PubMed Scopus (1685) Google Scholar, 5Seder R.A. Paul W.E. Davis M.M. Fazekas de St. Groth B. J. Exp. Med. 1992; 176: 1091-1098Crossref PubMed Scopus (942) Google Scholar, 6Hsieh C.S. Heimberger A.B. Gold J.S. O'Garra A. Murphy K.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6065-6069Crossref PubMed Scopus (723) Google Scholar, 7Shimoda K. Van Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1102) Google Scholar). 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 (8Ochensberger B. Rihs S. Brunner T. Dahinden C.A. Blood. 1995; 86: 4039-4049Crossref PubMed Google Scholar, 9Eglite S. Pluss K. Dahinden C.A. J. Immunol. 2000; 165: 2183-2189Crossref PubMed Scopus (53) Google Scholar). 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 (10Falcone F.H. Dahinden C.A. Gibbs B.F. Noll T. Amon U. Hebestreit H. Abrahamsen O. Klaucke J. Schlaak M. Haas H. Eur. J. Immunol. 1996; 26: 1147-1155Crossref PubMed Scopus (75) Google Scholar) but not to extracts of schistosomula. 2T. Brunner and C. A. Dahinden, unpublished data. 2T. Brunner and C. A. Dahinden, unpublished data. 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 (11Haisch K. Schramm G. Falcone F.H. Alexander C. Schlaak M. Haas H. Parasite Immunol. 2001; 23: 427-434Crossref PubMed Scopus (23) Google Scholar). 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 (12Doenhoff M.J. Pearson S. Dunne D.W. Bickle Q. Lucas S. Bain J. Musallam R. Hassounah O. Trans. R. Soc. Trop. Med. Hyg. 1981; 75: 41-53Abstract Full Text PDF PubMed Scopus (104) Google Scholar). SmEA was prepared by the methods of Boros and Warren (13Boros D.L. Warren K.S. J. Exp. Med. 1970; 132: 488-507Crossref PubMed Scopus (382) Google Scholar) and Carter and Colley (14Carter C.E. Colley D.G. J. Parasitol. 1978; 64: 285-290Crossref PubMed Scopus (46) Google Scholar) and was obtained from Dr. F. Yousif (Schistosome Biological Supply Program, Theodor Bilharz Research Institute, Imbaba, Egypt). Human basophils were purified by a previously described three-step procedure (15Haisch K. Gibbs B.F. Korber H. Ernst M. Grage-Griebenow E. Schlaak M. Haas H. J. Immunol. Methods. 1999; 226: 129-137Crossref PubMed Scopus (39) Google Scholar) 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 (16Brunner T. Heusser C.H. Dahinden C.A. J. Exp. Med. 1993; 177: 605-611Crossref PubMed Scopus (340) Google Scholar) and SmEA-mediated (10Falcone F.H. Dahinden C.A. Gibbs B.F. Noll T. Amon U. Hebestreit H. Abrahamsen O. Klaucke J. Schlaak M. Haas H. Eur. J. Immunol. 1996; 26: 1147-1155Crossref PubMed Scopus (75) Google Scholar) 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. 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. Proteins were separated by SDS-PAGE (12% T, 4% C) under reducing and nonreducing conditions according to Laemmli (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). For protein detection on the gels, silver staining was performed (18Heukeshoven J. Dernick R. Electrophoresis. 1988; 9: 28-32Crossref PubMed Scopus (640) Google Scholar). For blotting analysis, separated proteins were transferred onto nitrocellulose membrane (Schleicher & Schuell) via semidry blotting (30 min, 0.8 mA/cm2) (19Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2145) Google Scholar). Free binding sites were blocked by incubating the membrane with 0.05% (v/v) Tween 20 in The were incubated with alkaline or with antibodies the concentrations in containing 0.05% (v/v) Tween and binding was by a of and in Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). IPSE was purified by and affinity 2 of SmEA in 20 were to a of proteins was using a from to and fractions were for IL-4-inducing 5 and for protein The was determined by SDS-PAGE by silver staining. containing IPSE were and and for affinity the was to 50 by using For affinity was to (Amersham Biosciences) according to the manufacturer's of fractions and was with in 50 containing IPSE were by and stored at of the 20 of the purified IPSE was performed on an at was from mansoni eggs using the according to the manufacturer's of were obtained and used for of a cDNA library in by of the library according to the manufacturer's protocol. The cDNA library of S. mansoni eggs and a of The was as previously described Biochem. Parasitol. 1995; 75: PubMed Scopus Google Scholar). library has used for the of egg stage expressed sequence Parasitol. 2001; Full Text Full Text PDF Scopus Google Scholar). For of the cDNA a was with using the The was via an and were performed as described by and J. D.W. Scholar). was at °C in to detection of were for 5 at with and for at °C with were and the containing the were obtained by in vivo using the helper of was performed by using the on an IPSE was expressed as a tag protein in E. cDNA for IPSE was into the expression for and a for For this was performed with containing an and the following sites for the for the respectively. for were as for at for at and for at °C (30 and of J. D.W. Scholar). E. containing the recombinant were at 37 °C to of and expression of was by for an cells were by and in 50 0.8 was as after the cells by a a of and at The was with 20 and with 20 and in 50 was purified by affinity under conditions according to the manufacturer's protocol. the Research conditions for of the were purified was by at a of to and a such as phosphate-buffered saline antibodies and to were by containing of in 100 μl of saline and μl of were to The was after of antigen with The further 2 and the injection of IPSE were in saline to IPSE was by a after the the were and blood was for The was performed according to a procedure using the cell as of with S. mansoni previously were incubated with the culture or with μg/ml in or the were incubated for h with antibodies by staining with and were according to data were with (Amersham The data was for using the and protein were performed using the on the at The sequence of IPSE was to the data and has The for binding the IL-4-inducing to the was determined in all was by the the of proteins were in the was for the these the of the IL-4-inducing was to the fractions only a of the protein a and the IL-4-inducing to a SDS-PAGE analysis and silver staining of fractions of SmEA were performed under nonreducing a at that to about 20 under reducing The staining of the with the IL-4-inducing of the active fractions of the IL-4-inducing using of of SmEA separated by SDS-PAGE a and (11Haisch K. Schramm G. Falcone F.H. Alexander C. Schlaak M. Haas H. Parasite Immunol. 2001; 23: 427-434Crossref PubMed Scopus (23) Google Scholar), further suggesting that the or of the bioactive that the active principle is a protein of to (11Haisch K. Schramm G. Falcone F.H. Alexander C. Schlaak M. Haas H. Parasite Immunol. 2001; 23: 427-434Crossref PubMed Scopus (23) Google Scholar). Since schistosome extracts of Schistosoma and The Scholar), we whether the active principle is a glycoprotein be purified by affinity this active fractions from were separated by onto nitrocellulose and incubated with a of with various Indeed, to the active of for affinity since it only with of that in the active fractions and was also present in the fractions not this only the active fractions were for further affinity the fractions were the fractions with IL-4-inducing and SDS-PAGE by silver staining or by blotting with revealed that the fractions only the that in with the IL-4-inducing of the The fractions not by a high purity of the the active principle is a glycoprotein of containing to the at were not of the fractions from revealed an sequence with for the 20 suggesting that the of the in and not be to a with the of as by the IPSE cDNA not any with in the with the of data from an S. mansoni egg cDNA an of with the sequence and identity for the S. mansoni egg cDNA and were via assay by for was as a of was from The sequence of analysis revealed an of with a for a sequence of 20 The protein of sites at and and all 20 of the of the protein the sequence determined by of the purified natural the protein and data there was a sequence identity of with the sequence but that proteins not The of the active principle from the sequence was which is far the of the purified at that IPSE cDNA the active principle, recombinant IPSE was expressed in E. as a tag was from under conditions in and purified via affinity The protein was by the to and was performed at a protein concentrations to protein to assessed by SDS-PAGE under nonreducing that be into under reducing conditions on human basophils, a pronounced IL-4-inducing and was in this to natural The at or and a that is to that of The identity of the recombinant protein with natural IPSE was further by the following was with both recombinant and natural IPSE but showed to of SmEA basophils were with SmEA or not with the of a IL-4 production Finally, immunodepletion using the of to protein 100 μl of as obtained from the the IL-4-inducing from SmEA In contrast, the of normal as a not our data demonstrate that the recombinant to SmEA leads to a activation of basophils and release (10Falcone F.H. Dahinden C.A. Gibbs B.F. Noll T. Amon U. Hebestreit H. Abrahamsen O. Klaucke J. Schlaak M. Haas H. Eur. J. Immunol. 1996; 26: 1147-1155Crossref PubMed Scopus (75) Google Scholar). whether all of these are to basophils were incubated with SmEA and recombinant in and to and from were of to the IPSE at a purity of at Degranulation was observed basophils were with SmEA or recombinant IPSE but not following with or basophils considerable cell obviously to to and recombinant SmEA and of histamine, IL-4, and and 2 not any that the production of IL-13 was in the of IL-3 the cytokine considerable production of IL-13. with the of IL-4 these that IPSE is the only bioactive component in SmEA that basophils for and expression of studies that IgE has to be present on basophils for IPSE to whether IPSE as an IgE-binding factor, blotting were was to transferred to nitrocellulose and incubated with various concentrations of human Subsequent with revealed that IgE was by were obtained IgE was incubated with increasing amounts of these T. and G. unpublished data. these that IPSE is an IgE-binding We observed that IL-4-inducing is from schistosome eggs at 37 °C in culture medium (11Haisch K. Schramm G. Falcone F.H. Alexander C. Schlaak M. Haas H. Parasite Immunol. 2001; 23: 427-434Crossref PubMed Scopus (23) Google Scholar), suggesting that IPSE is a secreted further this of IPSE was performed in of using the the subshell area of schistosome eggs and to a of cells both and/or the subshell area in eggs that were by cells with the cells not or In contrast, the eggs in large with cells that during IPSE is and by were obtained with a to natural the the antibodies not to cells or the egg there was binding of normal and/or the to schistosome eggs or cells In with these R. B. R.A. 2001; PubMed Scopus Google Scholar) that an excretory/secretory from schistosome eggs the subshell we conclude that IPSE is enriched in the subshell area of the schistosome egg from which it is secreted into the interacting with a of cells by of Here we the sequence analysis, and recombinant expression of a glycoprotein secreted from S. mansoni IPSE basophils from naive human donors to rapidly release and IL-4 and the key cytokines a Th2-type immune response and IgE The for IPSE was a combination of and affinity with the The purified a high and of of about as assessed by nonreducing The purity of the natural bioactive was further by a preparation containing the which revealed suggesting that both of the was by the that all obtained from the were For the a was to a of that was the sequence of purified IPSE and the S. mansoni from the data the cDNA to the also However, the that from to IPSE were that the sequence a of this S. mansoni not or is expressed in the egg stage only at a very The protein of without sequence with on the at that the IPSE a of a IPSE may with the and an to this was also obtained by using the and on the at assessed by SDS-PAGE analysis performed under reducing and nonreducing recombinant IPSE with the of an of natural IPSE was to SDS-PAGE under reducing from to 20 a of for recombinant natural IPSE to be a glycoprotein with a of about that the recombinant protein to IPSE from natural the recombinant of basophils and release of histamine, IL-4, and IL-13. The high of at was in the as that of the natural the protein natural activation with SmEA and recombinant that activation by SmEA was to IPSE and not to of In with these of basophils with the of IPSE and not the antibodies were with natural IPSE and as well as natural IPSE from IPSE to be a the of in fractions and under reducing and nonreducing conditions as well as that IPSE be with antigen in S. mansoni eggs D.W. M.J. 1991; PubMed Scopus Google Scholar). glycoprotein is a antigen for of S. mansoni but thus far not further Indeed, blotting of antigen and with and antibodies as well as of antigen both to be Schramm and M. J. unpublished data. It has that of SmEA Th1 P. Harn D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) and are also for Th2 The was in a of with SmEA with the to induce IL-4, and IgE production M. Satoskar A.R. Nishizaki K. Abe M. Harn Jr., D.A. J. Immunol. 1999; Google Scholar). a component of SmEA that contains has to as a in to human M. Satoskar A.R. Nishizaki K. Harn Jr., D.A. J. Immunol. 2001; PubMed Scopus Google Scholar). Since natural IPSE to a for H. J. PubMed Scopus Google Scholar) and for T. T. T. Soc. 2001; PubMed Scopus Google Scholar), it is that natural IPSE contains However, for the of IPSE are not since recombinant IPSE has a powerful IL-4-inducing to human not human basophils a μg/ml using data not our not the that can to a Th2 by mechanisms cell basophils. Regarding the mechanism by which IPSE basophils, IgE and revealed that IgE has to be present on the cells for IPSE to (10Falcone F.H. Dahinden C.A. Gibbs B.F. Noll T. Amon U. Hebestreit H. Abrahamsen O. Klaucke J. Schlaak M. Haas H. Eur. J. Immunol. 1996; 26: 1147-1155Crossref PubMed Scopus (75) Google Scholar, K. Schramm G. Falcone F.H. Alexander C. Schlaak M. Haas H. Parasite Immunol. 2001; 23: 427-434Crossref PubMed Scopus (23) Google Scholar), suggesting that this via cross-linking of Fcε is by our blotting and binding of to IgE IPSE is an IgE-binding that basophils via cross-linking receptor-bound The type of IPSE and IgE is not it be an or cell binding is since the basophils were from nonsensitized in the of the recombinant were not The that IPSE as a be but sequence not show any with or can basophils H. Falcone F.H. Schramm G. K. Gibbs B.F. Klaucke J. M. Schlaak M. Eur. J. Immunol. 1999; PubMed Google Scholar), the concentrations are in the μg/ml In contrast, the that IPSE is a cell cell such as protein and protein A. G. J. Immunol. Google G. A. G. J. Immunol. 2000; PubMed Scopus Google can to IgE as can basophils, in the release of IL-4 and IL-13. release of IL-4 can be in the of increasing concentrations of human contains various and of may with of IgE for binding to these are under The in vivo of IPSE are not The pronounced IL-4 and IL-13 production by this in that IPSE be involved in and/or a Th2 response. for only of human blood G. and Scholar), to IL-4 during the h after antigen activation G. B. C. J. Immunol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and are to be an initial source of IL-4 (4Paul W.E. Seder R.A. Cell. 1994; 76: 241-251Abstract Full Text PDF PubMed Scopus (1685) Google Scholar, T. Heusser C.H. Dahinden C.A. J. Exp. Med. 1993; 177: 605-611Crossref PubMed Scopus (340) Google Scholar, S. Immunol. 1992; Full Text PDF PubMed Scopus Google Scholar, H. Falcone F.H. Schramm G. K. Gibbs B.F. M. A. Schlaak M. Immunol. 1999; PubMed Scopus Google Scholar, G. G. M. A. A. Immunol. 2000; PubMed Google Scholar). are blood cells and can sites of with may thus be to sites of schistosome egg basophils are observed in schistosome egg but in of the powerful of it is that basophils in this are and thus are not by staining. basophils with such as and Immunol. 2001; PubMed Scopus Google Scholar, S. A. E. M. C. J. Immunol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. B. P. S. P. M. Dahinden C.A. J. PubMed Scopus Google Scholar), which are by the and and the and suggesting that be important in the immune which is by for Since IPSE is secreted by schistosome as both from in egg culture supernatants and from the of IPSE on and in cells, this obviously also with basophils to the schistosome egg In IPSE is a parasitic glycoprotein with a and on human basophils. it can rapidly trigger basophils from nonsensitized human donors to release considerable amounts of IL-4, IPSE is a for biasing the immune response toward the Th2 phenotype in the of schistosome We are to A. M. G. U. T. and for and/or and to and S. for