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Inhibition of C3 covalent binding to IgG immune aggregates by recombinant protein G (domain III)
Complement activation, function and receptors
23 June 1997 - Poster presentations
receptor tyrceine kinase and its @and, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). in B cell adhesion and dffferentiation. By performing FACS analysis and immunohistochemistry we observed that c-Met is predominantiy expressed on CD38+CD77+ tonsiiiar B cells localized in the dark zone of the GC (centmblasts). On tonsil B cells, ligation of CD40 by CD40-iigand, induces a transient stmng .upregulation of expression of the -c-Met tymsine kinase, as shown by Western blotting. Stimulation of c-Met wtth HGF/SF leads to receptor phosphorylation and, in-addition, to enhanced integrtn-mediated adhesion of B cells to both VCAM-1 and fibmnectfn. Importantly, ELISA and RT-PCR analysis showed that the c-Met ligand, HGF/SF. is produced at high levels by tonsillar stmmal cells thus providing signals for the regulation of adhesion and migratlon within the iymphoid microenvironment. In conclusion, we have identifiid the c-Met-HGF/SF pathway as a novel paracrine signailing pathway regulating B ceil adhesion.
P.3.02.14
Redistribution of surface molecules on germinal center B ceils after bindina to foiiicuiar dendritic C&IS
Ernst Lindhout, Comelis de Grcot. Laboratory for Cell Bidogy and Histology, Academic Medkxl Center University of Amsterdam, Meibergdmef 75, Amsterdam, The Netherlands Within germinal centers (GCs) germinal center B cells (GC B cells) are found in dose contact with foiiicular dendrttic ceils (FDCs). Binding of B ceils to FDCs results in a rapid inhibition of apoptosis in GC B ceils. The precise mechanism of the rescue signal is unclear. It is known that FDCs and GC B ceils interact via several membrane molecules among which are interactions via the adhesion molecules ICAM-l/LFA-I and VCAM-lNLA-4, and interaction between immune complexes on FDCs and the B cell receptor (BCR) on B cells. For all these interaction pathways it has been demonstrated that they may postpone apoptosis in B cells to some extent. In the present study, we have investigated the precise localization of several different membrane molecules known to participate in FDC-B cells interactions using SEM, TEM and CLSM. Obsenration of FDC-B cell clusters showed a remarkable redistribution of membrane molecules: BCR, CDIQ, CD21, CD22, and CDlla tend to locate towards the contact area, whereas CD20 and CD40 did not. These data indicate that FDC-mediated rescue of GC B cells invoives interactions between a number of different membrane molecules.
1P.3.02.151
induction of antigen-specific isotype switching by In vitro immunization of human naive B lymph-
09:0&18:30/12:00-1400
P.3.03
P.3.03.01 -
109
Forum lounges
Complement activation, function and receptors Studies on the interaction between human C5a and ita receptor
P.N. Monk I, M.D. Barker’, J.E. Pease ‘, T. Crass *, W. Bautsch 2. ’ Krebs Institute Ibr Biomolecutar research, University of Sheffield, Sheffield, UK, * Medzinsiche Mikrobiologie, Medizinische Hochschule, Hannover, Germany
Introduction:The binding of the leukocyte chemoattractant, C5a (or its less active metabolite, C5a des Arg74), to its ceil surface receptor is still not fully understood, but is known to involve multiple contact sites. The N-terminus of the receptor binds C5a with high affinity, but is not invotved in the transduction of the iigand binding signal. in contrast, residues in the second extracellular loop (GIu’~ and Argsos)appear to be primarily responsible for receptor activation. Materials and Methods: We have developed a model system for the analysis of human C5a receptor activation, by expression in the rat basophilic ieukaemia cell line, RBL-2H3. Cells transfected with the receptor for human C5a were selected by two rounds of FACS. Human C5a receptor-bearing RBL cells undergo a pertussis toxin-sensftive exocytotic response to human C5a, determined as release of 3H-5-hydroxytryptamine. Reeultr: We have mutated a key residue, Lysm, at the C-terminus of C5a and also its putative counter-ion, Glulgg on the C5a receptor, to anatyse the relative contribution made by this interaction to iigand binding and receptor activation. We have found that mutation of Lyses to Glu in intact C5a has little effect on receptor binding or activation. In contrast, Lysss+Glum in C5a des Arg74 profoundly decreases receptor activation but not binding affinity. The activity of this mutant of C5a des Arg74 can be partially rescued by making the reciprocal receptor mutant, i.e. Glr~‘~+Lys’~. Similar results are obtained using short peptide analogues of the C-terminus of C5a and C5a des Arg74. Conclusion: It appears that the interaction between Lysss and GIu’~~ is uncovered by the loss of the C-terminal Arg74 during the rapid proteoiytic degradation of C5a by serum carboxypeptidase B. Lysm mutants of C5a des Arg74 which interfere with receptor activation but not binding may therefore be useful in the design of inhibitors of C5a activity.
P.3.03.02
inhibition of C3 covalent binding to IgG immune aggregates by recombinant protein G (domain iii)
E. Andersson ‘, A. Zafimpoulos *, E. Krambovitis *, C.A.K. Borrebaeck ‘. ’ Department of Immunotechnolog): Lund Universiify.Sweden, 2Laboratory of Applied Biochemistry and Immunology Institute of Molecular Biology and Biotechnology, Greece
F. Vivanco 1.2,E. Mufloz ‘, L. Vidarte It*, C. Pastor’,*. ‘Dept./nmunok@a, Fundacidn Jimdnez Diaz, Madrid, Spain, 2Dept. Bioquimica. Universidad Compiutense, Madrid, Spain
Introduction:The use of In vitro immunization technology for the generation
introduction:There is a group of bacterial proteins which have the ability to
of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in viw primary immune response. The aim of this study was to develw a novel protocol permitting the generation of IgG antibody repertoires in vitro, starting with mature naive peripheral blood B-ceils from healthy blood donors. Materialand Methods:The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-I) and a T helper cell epitops (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetanus toxoid. (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. Results: None of the donors whidr were examined after primary immunization showed at any time an IgG anti-VI specific antibody response, while ail the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/Q). Conclusion:We here describe a detailed reproducible protocol for a twostep in vitro immunization, which yields isotype switched, antigen-specific human antibodies.
recognize the constant domains of antibodies. Protein G interacts with the Fc region of human or rabbit IgG and with the CHl domain of mouse IgG1. These am the two antibody regions interacting with C3b, as previously described. We have studied whether Protein G (domain Ill) is able to block the C3b covatent binding to IgG (from human, rabbit or mouse) immune complexes (IC). Materials81Methods:IC were prepared at equivalence. Recombinant pmtein G domain ill was expressed in E. Coii and purtfied by ion exchange chromatography on a Mono Cl column (FPLC). Recombinant antibodies were produced as previously described. C3b binding to IC in the presence of l-[‘4C]-icdoacetamide, was determined by the appearance of radioactive high molecular weight bands on SDS-PAGE and fluorography. Rerulk Using rabbit IgG to form the IC, protein G produced a remarkable inhibition of C3b binding. in contrast, no inhibition could be detected using the F(ab’)n fragment, indicating that protein G interferes with the C3b binding to the Fc region. To confirm these data, recombinant antibodies (s&b) devoid of CHl domain and including the Fc rabbit or human IgGl, were produced. These scAb retained the ability to covalently bind C3b as the complete antibodies. Protein G inhibited the C3b binding to IC formed with scab of either human or rabbit origin. C3b binding to IC formed with native mouse IgGl was not inhibited. In the X-ray crystal structure of protein G domain Ill-Fab complex the C3b and protein G binding sites (residues 125-147 and 209-216, respectively) are located on opposite faces of the CHl domain, not interfeling with each other. Conclusion: Taken together, these data indicate that protein G domain Ill inhibits the C3b covalent binding to the Fc region of IgG.
23 June 1997 - Poster presentations
receptor tyrceine kinase and its @and, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). in B cell adhesion and dffferentiation. By performing FACS analysis and immunohistochemistry we observed that c-Met is predominantiy expressed on CD38+CD77+ tonsiiiar B cells localized in the dark zone of the GC (centmblasts). On tonsil B cells, ligation of CD40 by CD40-iigand, induces a transient stmng .upregulation of expression of the -c-Met tymsine kinase, as shown by Western blotting. Stimulation of c-Met wtth HGF/SF leads to receptor phosphorylation and, in-addition, to enhanced integrtn-mediated adhesion of B cells to both VCAM-1 and fibmnectfn. Importantly, ELISA and RT-PCR analysis showed that the c-Met ligand, HGF/SF. is produced at high levels by tonsillar stmmal cells thus providing signals for the regulation of adhesion and migratlon within the iymphoid microenvironment. In conclusion, we have identifiid the c-Met-HGF/SF pathway as a novel paracrine signailing pathway regulating B ceil adhesion.
P.3.02.14
Redistribution of surface molecules on germinal center B ceils after bindina to foiiicuiar dendritic C&IS
Ernst Lindhout, Comelis de Grcot. Laboratory for Cell Bidogy and Histology, Academic Medkxl Center University of Amsterdam, Meibergdmef 75, Amsterdam, The Netherlands Within germinal centers (GCs) germinal center B cells (GC B cells) are found in dose contact with foiiicular dendrttic ceils (FDCs). Binding of B ceils to FDCs results in a rapid inhibition of apoptosis in GC B ceils. The precise mechanism of the rescue signal is unclear. It is known that FDCs and GC B ceils interact via several membrane molecules among which are interactions via the adhesion molecules ICAM-l/LFA-I and VCAM-lNLA-4, and interaction between immune complexes on FDCs and the B cell receptor (BCR) on B cells. For all these interaction pathways it has been demonstrated that they may postpone apoptosis in B cells to some extent. In the present study, we have investigated the precise localization of several different membrane molecules known to participate in FDC-B cells interactions using SEM, TEM and CLSM. Obsenration of FDC-B cell clusters showed a remarkable redistribution of membrane molecules: BCR, CDIQ, CD21, CD22, and CDlla tend to locate towards the contact area, whereas CD20 and CD40 did not. These data indicate that FDC-mediated rescue of GC B cells invoives interactions between a number of different membrane molecules.
1P.3.02.151
induction of antigen-specific isotype switching by In vitro immunization of human naive B lymph-
09:0&18:30/12:00-1400
P.3.03
P.3.03.01 -
109
Forum lounges
Complement activation, function and receptors Studies on the interaction between human C5a and ita receptor
P.N. Monk I, M.D. Barker’, J.E. Pease ‘, T. Crass *, W. Bautsch 2. ’ Krebs Institute Ibr Biomolecutar research, University of Sheffield, Sheffield, UK, * Medzinsiche Mikrobiologie, Medizinische Hochschule, Hannover, Germany
Introduction:The binding of the leukocyte chemoattractant, C5a (or its less active metabolite, C5a des Arg74), to its ceil surface receptor is still not fully understood, but is known to involve multiple contact sites. The N-terminus of the receptor binds C5a with high affinity, but is not invotved in the transduction of the iigand binding signal. in contrast, residues in the second extracellular loop (GIu’~ and Argsos)appear to be primarily responsible for receptor activation. Materials and Methods: We have developed a model system for the analysis of human C5a receptor activation, by expression in the rat basophilic ieukaemia cell line, RBL-2H3. Cells transfected with the receptor for human C5a were selected by two rounds of FACS. Human C5a receptor-bearing RBL cells undergo a pertussis toxin-sensftive exocytotic response to human C5a, determined as release of 3H-5-hydroxytryptamine. Reeultr: We have mutated a key residue, Lysm, at the C-terminus of C5a and also its putative counter-ion, Glulgg on the C5a receptor, to anatyse the relative contribution made by this interaction to iigand binding and receptor activation. We have found that mutation of Lyses to Glu in intact C5a has little effect on receptor binding or activation. In contrast, Lysss+Glum in C5a des Arg74 profoundly decreases receptor activation but not binding affinity. The activity of this mutant of C5a des Arg74 can be partially rescued by making the reciprocal receptor mutant, i.e. Glr~‘~+Lys’~. Similar results are obtained using short peptide analogues of the C-terminus of C5a and C5a des Arg74. Conclusion: It appears that the interaction between Lysss and GIu’~~ is uncovered by the loss of the C-terminal Arg74 during the rapid proteoiytic degradation of C5a by serum carboxypeptidase B. Lysm mutants of C5a des Arg74 which interfere with receptor activation but not binding may therefore be useful in the design of inhibitors of C5a activity.
P.3.03.02
inhibition of C3 covalent binding to IgG immune aggregates by recombinant protein G (domain iii)
E. Andersson ‘, A. Zafimpoulos *, E. Krambovitis *, C.A.K. Borrebaeck ‘. ’ Department of Immunotechnolog): Lund Universiify.Sweden, 2Laboratory of Applied Biochemistry and Immunology Institute of Molecular Biology and Biotechnology, Greece
F. Vivanco 1.2,E. Mufloz ‘, L. Vidarte It*, C. Pastor’,*. ‘Dept./nmunok@a, Fundacidn Jimdnez Diaz, Madrid, Spain, 2Dept. Bioquimica. Universidad Compiutense, Madrid, Spain
Introduction:The use of In vitro immunization technology for the generation
introduction:There is a group of bacterial proteins which have the ability to
of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in viw primary immune response. The aim of this study was to develw a novel protocol permitting the generation of IgG antibody repertoires in vitro, starting with mature naive peripheral blood B-ceils from healthy blood donors. Materialand Methods:The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-I) and a T helper cell epitops (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetanus toxoid. (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. Results: None of the donors whidr were examined after primary immunization showed at any time an IgG anti-VI specific antibody response, while ail the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/Q). Conclusion:We here describe a detailed reproducible protocol for a twostep in vitro immunization, which yields isotype switched, antigen-specific human antibodies.
recognize the constant domains of antibodies. Protein G interacts with the Fc region of human or rabbit IgG and with the CHl domain of mouse IgG1. These am the two antibody regions interacting with C3b, as previously described. We have studied whether Protein G (domain Ill) is able to block the C3b covatent binding to IgG (from human, rabbit or mouse) immune complexes (IC). Materials81Methods:IC were prepared at equivalence. Recombinant pmtein G domain ill was expressed in E. Coii and purtfied by ion exchange chromatography on a Mono Cl column (FPLC). Recombinant antibodies were produced as previously described. C3b binding to IC in the presence of l-[‘4C]-icdoacetamide, was determined by the appearance of radioactive high molecular weight bands on SDS-PAGE and fluorography. Rerulk Using rabbit IgG to form the IC, protein G produced a remarkable inhibition of C3b binding. in contrast, no inhibition could be detected using the F(ab’)n fragment, indicating that protein G interferes with the C3b binding to the Fc region. To confirm these data, recombinant antibodies (s&b) devoid of CHl domain and including the Fc rabbit or human IgGl, were produced. These scAb retained the ability to covalently bind C3b as the complete antibodies. Protein G inhibited the C3b binding to IC formed with scab of either human or rabbit origin. C3b binding to IC formed with native mouse IgGl was not inhibited. In the X-ray crystal structure of protein G domain Ill-Fab complex the C3b and protein G binding sites (residues 125-147 and 209-216, respectively) are located on opposite faces of the CHl domain, not interfeling with each other. Conclusion: Taken together, these data indicate that protein G domain Ill inhibits the C3b covalent binding to the Fc region of IgG.