- Email: [email protected]
The loop region Lys154-Leu165 of human FcϵRIα is involved in human IRE binding — Characterization by alanine substitution mutagenesis
398
Functions of Ig isotypes and their receptors
tfve burst. Moreover, during granuiocytic maturation, a constant decrease in CD49d/29 expression was observed. In contrast, the SWC3*SWC8- monocytic ceils were mainly CD14+ and expressed high levels of the adhesion molecules. These results define porcine haematopoletic differentiation and maturation stages, of use in both porcine studies and the pig model for human analyses. Conclusion: The analysis of the maturation-dependent expression of particular SWC and CD antigens on porcine BMHC provide, for the first time, the possibility to discriminate myeloid from non-myeloid lineages in porcine BMHC, as well as the potential myelomonocytic precursor, and different maturation stages within the granulocytic and monocytic lineages. In this context, the maturation-dependent adhesion molecule expression on porcine myeloid BMHC was found to be similar to the human, which would infer common roles of functionality.
09:09-18:30/12:00-14:W
Forum
P.3.06
Functions of Ig isotypes and their receptors
P.3.06.01
Opsonic and bactericidal activity induced by different IgG subclass antibodies after immunisation with menlngococcal group B outer membrane vesicle vaccine
A. Aase, E.A. Heiby ‘, T.E. Michaelsen. Department of Vaccine/logy,National InstiMe of Public Health, Oslo, Not-wax ’ Department of Sactedotog~ National Institute of Public Health Oslo, Norway
Introduction:The immune response against meningococci after vaccination with the Norwegian meningococcal outer membrane vesicle (OMV) vaccine is dominated by IgGl and lgG3 antibodies. Occasionally a moderate lgG2 response is detected, whereas lgG4 is infrequently found. In this study we have analysed the opsonic and bactericidal activitv of ourffied laGI, laG2 and laG3 subclass ant&dies from vaccinee sera against meningoc&ci.’ Materials and Methods: Plasma IgG from one vaccinee with high IgGl and lgG3 responses against OMV was isolated using anion exchange chromatography. This IgG preparation was further separated on a Protein A column to get pure IgGl and lgG3 antibodies. From another vaccinee, with moderate IgGl and high lgG2 levels, IgG was isdated by anion exchange chromatography. The IgG pmpartion was further digested with papain in absence of any reduction reagent. Under these conditions lgG2 is papain resistant, whereas IgGl is sensitive and will be digested. Finally, intact lgG2 antibodies were isolated by gel filtration. The purity and quantity were measured in ELISA on OMV coated microplates. Opsonic activity was either measured as intemalfsation of FITC labelled meningococci (the vaccine strain) by neutrophils, or as generation of respiratory burst in neutrophils during phagocytosis. Opsonic activity was measured both in the presence and the absence of complement, and the responses were analysed by flow cytometry. Bactericidal activity was measured against the vaccine strain. Results: The opsonic activity in the absence of complement revealed IgGl and lgG3 to be highly active and lgG2 to be negative, whereas in the presence of complement all three subclasses were highly active. These results are in accordance with our previous studies using chime& IgG subclass antibodies. The activity of putified subclass antibody revealed similar activity to that of crude plasma. In the bactericidal assay, preliminary data indicate that IgGl is the most effective, but lgG2 and lgG3 also confer bactericidal activity. The epitope specificities of the different preparations have not yet been determined. Conclusion: Antibodies of IgGl, lgG2 and lgG3 subclasses against group B meningococci may all contribute to protection against meningococcal disease as they all show both opsonic and bactericidal activity. P.3.06.02
Is IgE-FcoRI bindlng and induction of target cell triggering a process involving different FCO domains and conformational rearrangement?
F. Krfcek’, C. Ruf ‘, A. Nechansky ’ , E. Pursch ’ , M.P. Rudolf *, B.M. Stadlers. ’ Novartis Forschungsinstitut GmbH, Vienna, Austria, 21nstituteof Immunology and Allergotog~ University Bern, Switzerland
Intmductlon:Binding of IgE to its high affinity receptor (FcsRI) is a crucial event in the pathogenesis of allergic reactions. Crosslinking of receptor-bound IgE by specific allergens triggers degranulation and modulates cytokine production of
25 June 1997 - Poster presentations
target cells (e.g. mast cells, basophils, eosinophils). IgE binds to FcoRl via the Ce3 domain in a 1:I complex. Conformational rearrangements within FCs have been proposed to accompany the binding process (1). Furthermore, though not being directly involved in receptor binding, amino acid sequences beloogfng to the C&4 domain of IgE have also been speculated to play a role in the induction of anaphylacbc reactions (2). Here we present experiments which support these speculations and provide tools to further evaluate this hypothesis. Materfals and Methods: Using bacterially produced recombinant C&3 as immunogen, moncclonal antibodies (mAb) were generated in mice. Receptor binding studies were done using recombinant soluble CC chain of FcsRI (FcsRIa), produced in insect cells. By in vitro expression of DNA encoding truncated C&3 fmgments the epitope regions for these antibodies and another anti C&3 mAb (BSWl7) were &e&&f. Screening of random peptfde phage display libraries by biopanning on BSW17 resulted in the identification of peptides (mimotooes) mimicking the authentic BSW17 epitope on Fc&. Phage &ides displaying BSWI 7 mimotopes were used to induce an anti human IgE immune response in rabbi. Raeulta: Anti C&3 mAb obtained from immunization with bacterially synthesized r&3 do not recognize native IgE in solution. However, IgE bound to Fc~Rla is by these antibodies indicating C&3 upon receptor IgE solution thereby inhibiting receptor attachment but also binds to Fc&Rla-bound IgE. However, BSWI 7 is non anaphylactogenic. Two types of mimotopes were identified from phage library screening: one mimicking the C&3 (378-375) region postulated to be involved in receptor binding, the other mimicking the CE~ (566-508) region speculated to be involved in target cell trfggerfng. Conclualons:1. High affinity binding of IgE to Fc~Rla is accompanied bv a structural rearrangement of the C&3 domain. 2. The (partially contradictoryj biological properties of BSW17 can be explained by the conformational epitope involving both a C&3 segment (FccRI binding inhibition) and a particular C&4 segment (recognition of receptor-bound IgE, yet non anaphyfactogeniclty). Anti BSW17 mimotope antibodies could be used to further investiaate the seauential events resulting in target cell triggering. [I] Beavil et al. Biochemistry 34: 1449 (1995). [2] Stanworth et al.; Lancet 336: 1279 (1996).
P.3.06.03
The loop region Ly~‘~-Leu’~ of human FcrRla is lmrolved In human IRE blnding - Characterization by alanlne substitution mutagenesis
A. Nechansky, F. Krfcek. NovartiS Forschungsinstitut GmbH, Brunnemtrasse 59. A-1235 Wien Vienna. Austria
Introductlcn:The interaction of human IgE with the cr-chain of the high afffnity receptor for IgE (FcsRkx) plays a pivotal role in allergy of type 1 (I). The knowledge of the contact residues involved in this reaction would be of great interest for designing low molecular weight inhibltors. Binding regions for IgE were identified in three - relatively large - independent regions of the u(2) domain in the extracellular pad of the receptor (2). We investigated the contribution to IgE binding of residues residing within a carboxy terminal loop region of this extracellular domain by replacing amino acids with alanines. Material and Methods:Mutagenesis was performed with PCR. Two Hindlll restriction sites flanking the loop region, were introduced in the receptor construct and were further used for cassette mutagenesis. Each recombinant plasmid was transiently expressed in COS-7 cells as amino terminal fusion to human serum albumin (HSA) and secreted into the medium. The HSA part was used for quantitation of recombinant receptor proteins in cuitum supematants and for detection of bound receptor to immobilized IgE in ELISA experiments. Addftionally, mAb 15/l (3), which inhibits binding of IgE to the receptor, was screened for binding to the mutated receptor proteins. Reauft~ Loop residue Ttp w6 is essential for binding of mAb 15/l whereas IgE binding is not affected at all when this tryptophan is substituted bv alanlne. Removal of the negatfve charges in the loop ‘neither effected IgE-nor 16/l binding. The Ser’@+Ala mutation msufted in a moderate reduction of IgE and 15/l binding whereas the additional amino acid substitution Glnw7+Ala abolished binding of both. Conclusion: Binding of human IgE to FmRla is partly mediated via residues in the receptor loop sequence Lysls+-LeulES.The mAb 15/l interacts dlnsctlv with this locp which contains an- essential tryptophan residue. Substftutfon of Ser’@, which is highly conserved among Fez and Fey receptors and may therefore represent a structural element, partially effects IgE and 15/l binding. [I] Gould et al., Nature 366: 421428, 1993 [2] Hulett et al., Eur. J. Immunol. 23: 640-645, 1993 [3] Wang, B., J. Exp. Med. 175, 135+1365, 1992
Functions of Ig isotypes and their receptors
tfve burst. Moreover, during granuiocytic maturation, a constant decrease in CD49d/29 expression was observed. In contrast, the SWC3*SWC8- monocytic ceils were mainly CD14+ and expressed high levels of the adhesion molecules. These results define porcine haematopoletic differentiation and maturation stages, of use in both porcine studies and the pig model for human analyses. Conclusion: The analysis of the maturation-dependent expression of particular SWC and CD antigens on porcine BMHC provide, for the first time, the possibility to discriminate myeloid from non-myeloid lineages in porcine BMHC, as well as the potential myelomonocytic precursor, and different maturation stages within the granulocytic and monocytic lineages. In this context, the maturation-dependent adhesion molecule expression on porcine myeloid BMHC was found to be similar to the human, which would infer common roles of functionality.
09:09-18:30/12:00-14:W
Forum
P.3.06
Functions of Ig isotypes and their receptors
P.3.06.01
Opsonic and bactericidal activity induced by different IgG subclass antibodies after immunisation with menlngococcal group B outer membrane vesicle vaccine
A. Aase, E.A. Heiby ‘, T.E. Michaelsen. Department of Vaccine/logy,National InstiMe of Public Health, Oslo, Not-wax ’ Department of Sactedotog~ National Institute of Public Health Oslo, Norway
Introduction:The immune response against meningococci after vaccination with the Norwegian meningococcal outer membrane vesicle (OMV) vaccine is dominated by IgGl and lgG3 antibodies. Occasionally a moderate lgG2 response is detected, whereas lgG4 is infrequently found. In this study we have analysed the opsonic and bactericidal activitv of ourffied laGI, laG2 and laG3 subclass ant&dies from vaccinee sera against meningoc&ci.’ Materials and Methods: Plasma IgG from one vaccinee with high IgGl and lgG3 responses against OMV was isolated using anion exchange chromatography. This IgG preparation was further separated on a Protein A column to get pure IgGl and lgG3 antibodies. From another vaccinee, with moderate IgGl and high lgG2 levels, IgG was isdated by anion exchange chromatography. The IgG pmpartion was further digested with papain in absence of any reduction reagent. Under these conditions lgG2 is papain resistant, whereas IgGl is sensitive and will be digested. Finally, intact lgG2 antibodies were isolated by gel filtration. The purity and quantity were measured in ELISA on OMV coated microplates. Opsonic activity was either measured as intemalfsation of FITC labelled meningococci (the vaccine strain) by neutrophils, or as generation of respiratory burst in neutrophils during phagocytosis. Opsonic activity was measured both in the presence and the absence of complement, and the responses were analysed by flow cytometry. Bactericidal activity was measured against the vaccine strain. Results: The opsonic activity in the absence of complement revealed IgGl and lgG3 to be highly active and lgG2 to be negative, whereas in the presence of complement all three subclasses were highly active. These results are in accordance with our previous studies using chime& IgG subclass antibodies. The activity of putified subclass antibody revealed similar activity to that of crude plasma. In the bactericidal assay, preliminary data indicate that IgGl is the most effective, but lgG2 and lgG3 also confer bactericidal activity. The epitope specificities of the different preparations have not yet been determined. Conclusion: Antibodies of IgGl, lgG2 and lgG3 subclasses against group B meningococci may all contribute to protection against meningococcal disease as they all show both opsonic and bactericidal activity. P.3.06.02
Is IgE-FcoRI bindlng and induction of target cell triggering a process involving different FCO domains and conformational rearrangement?
F. Krfcek’, C. Ruf ‘, A. Nechansky ’ , E. Pursch ’ , M.P. Rudolf *, B.M. Stadlers. ’ Novartis Forschungsinstitut GmbH, Vienna, Austria, 21nstituteof Immunology and Allergotog~ University Bern, Switzerland
Intmductlon:Binding of IgE to its high affinity receptor (FcsRI) is a crucial event in the pathogenesis of allergic reactions. Crosslinking of receptor-bound IgE by specific allergens triggers degranulation and modulates cytokine production of
25 June 1997 - Poster presentations
target cells (e.g. mast cells, basophils, eosinophils). IgE binds to FcoRl via the Ce3 domain in a 1:I complex. Conformational rearrangements within FCs have been proposed to accompany the binding process (1). Furthermore, though not being directly involved in receptor binding, amino acid sequences beloogfng to the C&4 domain of IgE have also been speculated to play a role in the induction of anaphylacbc reactions (2). Here we present experiments which support these speculations and provide tools to further evaluate this hypothesis. Materfals and Methods: Using bacterially produced recombinant C&3 as immunogen, moncclonal antibodies (mAb) were generated in mice. Receptor binding studies were done using recombinant soluble CC chain of FcsRI (FcsRIa), produced in insect cells. By in vitro expression of DNA encoding truncated C&3 fmgments the epitope regions for these antibodies and another anti C&3 mAb (BSWl7) were &e&&f. Screening of random peptfde phage display libraries by biopanning on BSW17 resulted in the identification of peptides (mimotooes) mimicking the authentic BSW17 epitope on Fc&. Phage &ides displaying BSWI 7 mimotopes were used to induce an anti human IgE immune response in rabbi. Raeulta: Anti C&3 mAb obtained from immunization with bacterially synthesized r&3 do not recognize native IgE in solution. However, IgE bound to Fc~Rla is by these antibodies indicating C&3 upon receptor IgE solution thereby inhibiting receptor attachment but also binds to Fc&Rla-bound IgE. However, BSWI 7 is non anaphylactogenic. Two types of mimotopes were identified from phage library screening: one mimicking the C&3 (378-375) region postulated to be involved in receptor binding, the other mimicking the CE~ (566-508) region speculated to be involved in target cell trfggerfng. Conclualons:1. High affinity binding of IgE to Fc~Rla is accompanied bv a structural rearrangement of the C&3 domain. 2. The (partially contradictoryj biological properties of BSW17 can be explained by the conformational epitope involving both a C&3 segment (FccRI binding inhibition) and a particular C&4 segment (recognition of receptor-bound IgE, yet non anaphyfactogeniclty). Anti BSW17 mimotope antibodies could be used to further investiaate the seauential events resulting in target cell triggering. [I] Beavil et al. Biochemistry 34: 1449 (1995). [2] Stanworth et al.; Lancet 336: 1279 (1996).
P.3.06.03
The loop region Ly~‘~-Leu’~ of human FcrRla is lmrolved In human IRE blnding - Characterization by alanlne substitution mutagenesis
A. Nechansky, F. Krfcek. NovartiS Forschungsinstitut GmbH, Brunnemtrasse 59. A-1235 Wien Vienna. Austria
Introductlcn:The interaction of human IgE with the cr-chain of the high afffnity receptor for IgE (FcsRkx) plays a pivotal role in allergy of type 1 (I). The knowledge of the contact residues involved in this reaction would be of great interest for designing low molecular weight inhibltors. Binding regions for IgE were identified in three - relatively large - independent regions of the u(2) domain in the extracellular pad of the receptor (2). We investigated the contribution to IgE binding of residues residing within a carboxy terminal loop region of this extracellular domain by replacing amino acids with alanines. Material and Methods:Mutagenesis was performed with PCR. Two Hindlll restriction sites flanking the loop region, were introduced in the receptor construct and were further used for cassette mutagenesis. Each recombinant plasmid was transiently expressed in COS-7 cells as amino terminal fusion to human serum albumin (HSA) and secreted into the medium. The HSA part was used for quantitation of recombinant receptor proteins in cuitum supematants and for detection of bound receptor to immobilized IgE in ELISA experiments. Addftionally, mAb 15/l (3), which inhibits binding of IgE to the receptor, was screened for binding to the mutated receptor proteins. Reauft~ Loop residue Ttp w6 is essential for binding of mAb 15/l whereas IgE binding is not affected at all when this tryptophan is substituted bv alanlne. Removal of the negatfve charges in the loop ‘neither effected IgE-nor 16/l binding. The Ser’@+Ala mutation msufted in a moderate reduction of IgE and 15/l binding whereas the additional amino acid substitution Glnw7+Ala abolished binding of both. Conclusion: Binding of human IgE to FmRla is partly mediated via residues in the receptor loop sequence Lysls+-LeulES.The mAb 15/l interacts dlnsctlv with this locp which contains an- essential tryptophan residue. Substftutfon of Ser’@, which is highly conserved among Fez and Fey receptors and may therefore represent a structural element, partially effects IgE and 15/l binding. [I] Gould et al., Nature 366: 421428, 1993 [2] Hulett et al., Eur. J. Immunol. 23: 640-645, 1993 [3] Wang, B., J. Exp. Med. 175, 135+1365, 1992