- Email: [email protected]
Molecular analysis of the function of the human IgA Fc domain
402
Functions
were further anatysed by restriction mapping and sequencing. Expression levels were measuredby FACS analysis of cDNAs transfected in L293 fibroblasts. Reaultsz In the present study, a total of nine FcyRIII-A cDNA clones obtained from two independent NK and LAK cell libraries were analysed. Different types of FcyRIII-A cDNAs were recognized representing four distinct groups of transcripts (al, a2,a4, AEC2) which either contained unique 5-UT sequences due to alternative promoter usage (al, a2, a4), or diverging sequences after the EC1 exon (a4), or were generated by alternative splicing (AECP). The al and a2 transcripts appeared to encode the normal FcyRIII-A receptor. Transfection experiments showed that surface expression of al, a2,and AEC2 is dependent on the FcR y subunit. Fcy FtIII-Aa4/FcR y subunit transfectants were incapable to express any functional Fey Rlll variant on 293 fibroblasts. Different patterns of anti-FcyRIII mAb reactivities were observed for al and a2 compared to AEC2 allowing for the identification of attemative spliced FcyRIII-A gene products on Fey RIII-expressingcells. Con&don: These date indicate. that in addition to the gene duplication event generating the two FcyRIII-A and FcyRIII-B receptors alternative splicing serve to control for the expression of modiiied Fey Rlll receptors. Further studies on the function and tissue distribution are necessary to elucidate the biological significance of such modified Fc receptors.
P.3.06.17
Noo;;;:
as promoter of metallolectin oxidative
A.J. Kulberg, J.B. Berkun. N.F: Gamaieya Epidemiology and Microbiology Institute, Moscow, Russia Introduction: Metalloproteins, like for example ConA, may exist in nonoxidized form if only transition metal(s) is located In nonpolar space of the protein molecule. It suggests that the conformational transitions of the metallolectin molecule resulting hydratation of the metallocanying space would be accompanied by the protein oxidatlve damage(s). To induce such a process we decided to use for ConA treatment the preparation of serum IgG because of amphypatic characteristics for several portions of its molecule (A.J. Kulberg et al., 1960-l 996). Msterlals and Mahods: Commercial preparations of ConA, human IgG and human serum albumin (control) were mixed in different proporttons followed by testing hemaggluttnation activity of ConA against human erythrocytes, U.V. analysis of the preparations tested and their molecular characteristics by ultrafiltration technique. Resulte: Adding IgG in over increasing concentrations to ConA results in rising inhibition of the ConA hemagglutination activity. The effect of serum albumin upon the ConA activity was negligible. Equimolar mixture of ConA and IgG versus ConA and IgG tested separately shows decrease in U.V. absorption, predominantly between 210-240 nm. The phenomenon of hypochromy doesn’t accompany any cleavage of the ConA-IgG mixture. However, when the above mixture is irradiated additionally wlthin minutes at 260 nm a clearly detected cleavage of the proteins (predominantly ConA) doss occur. Conclusion: Interaction ConA with IgG provokes change in ConA conformation revealing in U.V. spectral characteristics and loss of hemagglutination activity. The state of the complex formed can be considered as an intermediate for the further protooxidative cleavage of metallolectin and IgG. It may imply that the biological activity of lectins is realized through activation of their oxidative potency mediated by transition metal(s) co-ordinated by these proteins.
1 P.3.06.16
25 June 1997 - Poster presentations
of Zg isotypes ana’ their receptors
1 IgG-mediated anaphylaxis and inflammation are Impaired in mice lacking FcyRlll
W.L.W. Hazenbos’, J.E. Gessner*, F.M.A. Hofhuis’, H. Kuipers’, D. Meyers, I.A.F.M. Heijnen’, R.E. Schmidt2, M. Sandor3, P.J.A. CapeI’, M. Da&on4, J.G.J. van de Winkel ‘, J.S. Verbeek’. ’ Dept. oftmmunology, University Hospital lltrecht, The Nethettands, 2Dept. of Clinical Immunolo~ Me&al School, Hannover, Germany 3fatho/ogy and Lab. Medicine, University of Wisconsin, USA, 4Lab. d’lmmunologie Cellulaire et Clinique, Inst. Curie, Paris Extensive in vitro studies have demonstrated that various IgG-mediated effector functions of the immune system are triggered by interaction between antigenbound IgG and receptors for IgG (FcyR). In order to study the contribution of FcyR to in vivo immune reactions, we have generated mice deficient in the ligand binding IYchain of FcyRIII. Macrophages from these mice failed to bind and ingest particles opsonized with IgGi, in contrast to IgG2a or IgGPb. indicating an apparent specificity of IgGl for FcyRIII, the in vivo relevance of which is currently under further investigation. IgGtriggered in vitro degranulation of mast cells, and in vivo passive cutaneous anaphytaxis were abrogated in FcyRIII-‘- mice, while both reactions developed normally after triggering with IgE. In addition, after being depleted of complement, FcyRIII-‘- mice did not elidt an Arthus reaction, the classical model for IgG-mediated inflammation. In sum, FcyRlll is essential for recognition of complexed IgGl and for IgG-mediated anaphylaxis and complement-independent inflammation. These findings indicate a therapeutic potential for interfering in the function of FeyRIII.
P.3.06.19
FcyRll slgnal transduction in neutrophlls is differently regulated by receptor-type protein-tyroslne phosphatases CD45 and CD148 (HPTPr$DEP-1)
M. Hundt, R.E. Schmidt. Department of Clinical Immunology Hannover Medical School, Germany Introduction: Activation of the 40 kDa low-affinity receptor for IgG (FcyRII, CD32) leads to tyrosine phosphorylation, mobilisation of intracellular calcium and production of superoxide anions in neutrophils. It has been established that protein-tyrosine kinases and phosphatases am essential for the regulation of Intracellular signatllng. CD45 is a type I receptor-type protein-tyrosine phosphatase (RPTP) with two enzymatically catalytic regions. Recently it has been demonstrated that co-cross-linking of CD45 modulates the signal transduction pathway of FcyRll in neutrophils. In contrast, the functional characteristics of CD146, a type Ill RPTP wtth only one phosphatase domain, is unknown. Mstsrlals and Mahods: The expression of CD45 and CD146 was analysed by indirect immunoftuorescence with saturating amounts of mouse monoclonal antibodies (mAb) of the IgGI isotype: ICO-166 (isotype controt), 7E.12 (CD45), and 143-41 (CD146). The rise of free intracellular calcium was measured by flow cytometry using fluo-3. Neutrophils were preincubatad with as&es of mAb at a dilution of 11100 and than washed with PBS. Subsequently FcyRll was cross-linked by mAb IV.3 (IgGsb, ascites, l/600) and F(ab’)s goat anti-mouse (200 &ml). The production of superoxide anions was measured by lucigenin enhanced chemiluminescence after idenfcal pretreatment as described above. Rssulta: Neutrophils express CD46 in higher density than CD146. Co-crosslinking of CD45 with FeyRll reduced calcium mobilisatlon and respiratory burst significantly in comparison to control ascites. In wntrast, prelncubatlon with CD146 decreased superoxide anion generation, but did not change calcium rise: Meanf SEM,N GAMonly ICO-16s
Expression Calcium mobilisation
MFI, 3 MCF, 6
7E.12
(isotvpe contron
(CD4w
14341 (CDW)
.I. 29.2f1.4
7.0zt3.2 54.4fl.7
169.3f65.4 29.9f2.4
52.0fll.3 59.7f3.4
115f66
316f230
146f93
152f69
Superoxide anion generation
counts(x103),3
Conclusion: Bothclustered human RPTP, CD45 and CD146, inhibit FcyRlI induced superoxide anion generation, but they diier in regulation of calcium mobilisation. In previous studies it has been demonstrated that Fey RII induced Hz4 production is only in part dependent on calcium as a second messenger. Therefore, it is suggested that co-cross-linking of FcyRll and CD45 inhibits superoxide anion generation first by calcium independent pathways, second upstream, and possibly third downstream of calcium release in calcium dependent pathways. In contrast, CD146 has to dephosphorylate proteins downstream, but not upstream of calcium mobilisation. These distinct functional capacities may be important for differential regulation by currently unknown ligands of CD45 and CD146 (This work was supported by the Deutsche Forschungsgesellschaft Hu 704/1-l and Sonderforschungsbereich 244/AO9.)
P.3.06.20
Molecular analysis of the function of the human IgA Fc domain
J.M. Hexham, L.N. Carayannopoulos, J.D. Capm. Mo/ecu/ar lmmunofogy Center, Dept MicrobioiogK UT Southwestern Medical Center at Dallas 6000 Harry Hines Blved, Da//as, TX, USA Introduction: IgA is the immunoglobulin subclass largely responsible for humoral immune protection at the exposed mucosal surface. However relatively liile is known about the effector functions of the IgA Fc region. We have used a panel of mutants to define the sites on IgA for the specific IgA Fc receptor binding and the polymeric immunoglobulin receptor binding. Materials and Methods: IgA/lgG domain swap mutants as well as point and truncation mutants of human IgAl were constructed and expressed as recombinant baculoviruses. Binding of these mutant IgA molecules was assayed by rosetting with the IgA specific Fc receptor expressed on the HL60 cell line. Binding to the polymeric immunoglobulin receptor was measured by FACS of the receptor-expressing cell line MDCK and by ELISA using baculovirus expressed poly Ig receptor. Resutk The IgA specific Fc receptor binds to IgA at the junction of the CH2 and CH3 domains, in a position analogous to the site used by Staphlococcal Protein A in its interaction with IgG. The poly lg receptor binds to dime& IgA in the CH3 domain. Binding is specific to dimetic IgA. The role of tailpiece and J chain, which are wmponents of the complex is presently under investigation. We are also attempting to minimise the binding domain to enable efficient targetting of lgkbased constructs to the mucosla surface.
Functions of Ig isotypes and their receptors
25 June 1997 - Poster presentations
Conclueions: The disection of the molecular functions of IgA and other antibodies will enable the construction of antibodies with enhanced or modffed effector functions. In particular the poly lg receptor binding motif may be used to target antibodies or drugs to mucous membranes.
( P.3.06.21
1 Clinical value of soluble IgG Fc receptor type III (sFcyRIII) in plasma from patients with neutropenia
Harry R. Koene ‘, Masja de Haas ’ , Marion Kleijer ‘, Tom W.J. Huizinga *, Dirk Roes’, Albert E.G.Kr. von dem Borne ‘a3.’ Central Labomtory of the Netherfands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunolosv, Univemity of Amsterdam, Amsterdam, The Nethetlands, * Unfversfty Hospital Leiden, Department of Rheumatof~, Amsterdam, The Netherlands, 3Academic Medical Centre, Department of Hematol~, Amsterdam, The Netherlands Introductfon: Previous studies have shown that the plasma level of soluble IgG Fc receptor type Ill (sFcyRIII) is a measure of the total body neutrophil mass. The aim of this study was to determine whether the plasma level sFcy Rlll can bs used as a parameter to assess the risk of contracting bacterial infections for patients with neutropenia. Methods: We measured sFcyRlll levels in plasma of patients with neutropenla of unknown origin, whose blood was sent to our laboratory for diagnostic evaluation of neutropenia (neutrophil count < 1500@). Clinical data from 66 neutmpenic patients were obtained from the patient files. The occurrence or absence of bactedal infections was scored in the period of three months before to three months after a single sFcy Rlll measurement. In addition, longitudinal data were obtained from 21 patients. Reeultr: of the 66 included neutropenic patients, 15 had suffered from a bacterial infection. Neither the age distribution, nor the genotype frequencies of neutmphil FcyR polymorphisms were different among the group with and without infections. Both neutmphil count and plasma level sFcy Rlll were significantly lower in the patient group with infections, compared to the non-infected group (p = 0.03 and p < 0.0001, respectively). No infections were reported for patients who had plasma sFcyRlll levels above 100 arbiirary units (AU; N: 40-180). After matching each infected patient with two non-infected patients having the same neutrophil count, sFcy Rlll plasma levels remained significantly lower in the group with infections (p = 0.0001). For the patients who were followed in time (mean follow-up: 9 months), no infections were reported when sFcyRlll levels were above 100 AU. Conotuelon: A plasma sFcyRlll level above 100 AU in patients with neutmpenia might indicate a normal bone marmw neutmphil production and therefore no increased risk of contracting bacterial infections.
1P.3.06.22 1 Discovery of an adenosine deaminase activity in the preparates of human IgO3 Andrey V. Zavialov, V.P. Zav’yalov. lnstftute of /mmuno/ogica/ Engineering, 142380 Lyubuchany, Chekhov District, Moscow Region, Russia Introduction: Earlier by means of radio ligand binding assay we have shown that the immobilised IgG and Bence-Jones proteins bind pudne nucleotide monophosphates and nucleosides with high specific affinity (G = 2 x 1O-g M for cGMP and inosine. Kd = 8 x lo-’ M for CAMP, & = 6 x lo-’ M for AMP and GMP). Materialsand Methods: Human IgG was separated from the blood plasma of heaithy donors by the ion exchange chromatography. The lgG3 subclass was separated from other subclasses on the Sephamse Protein A column, and continuously treated by ion exchange chromatography on MonoC and gel filtration on Supemse 12 columns. Reeults: We discovered that the fractions of IgG from the blood plasma of healthy donors eluted from the ion exchange column possess an adenosine deaminase activity. More than 90% of the activity is related to the lgG3 subclass separated from other IgG subclasses on the Sephamse Protein A column and continuousiy treated by ion exchange chromatography on MonoC and gel filtration on Supemse 12 columns. The adenosine deaminase activity of lgG3 is characterfsed by Michaelis constants Ku = 2 + 3 x 1O-3 M and Km = 20 t 30 k-l. Involving activity disappears after modification of tryptophan residues or after reduction and alkylation of disulphide bonds in non-denatured conditions. It was found that the lgG3 inriches 20-fold of an adenosine deaminase activity after gel filtration in 8M urea and renaturation. Conclusion: The data described above permit to suggest that the hinge region of lgG3 provides access to the adenosine deaminase active site(s) located in the constant parts of Fab subunit.
P.3.06.23
403
Elucidation of cleavage site requirements for human IgAl hinge cleavage by etreptococcal IgAl PrOteaseS
MR. Batten, J.M. Woof. Department of Mofecufar and Cellular Pathology, University of Dundee Medical School, Ninewells Hospftal, Dundee, UK
Introduction:IgA is the major immunoglobulin isotype in mucosal secretions, with its main role being defence of the mucosae against bacterial and viral invasion. S-IgA is resistant to most pmteolytic degradation, with the exception of the IgAl proteases, produced by pathogenic bacteria responsible for diseases such as pneumonia, meningitis and periodontal disease. Cleavage of IgAl into Fab and Fc fragments results in loss of structural integdty and antibacterial function, facilitating mucosal colonisation and establishment of infection. Cleavage occurs at specific bonds within the IgAl hinge region, although the exact requirements for substrate recognition and cleavage have not been elucidated. This study aims to determine the precise structural requirements for IgAl cleavage by the IgAl pmteases. Materielsand Methods:Site-directed mutagenesis by PCR overlap extension was performed on a plasmid based on the mammalian expression vector, pEE6HCMV. The vector contains the IgAl heavy chain gene, permitting manipulation of the hinge region. Constructs were expressed in an established CHO cell expression system to produce chimeric anti-NIP human IgAl antibodies. Each antibody was incubated with log-phase bacterial cufture from three streptococcal strains, then immunoblotted with anti-IgA antibodies to assess cleavage susceptibility. Resutts: Three mutants were analysed in the study. These have Thr 226 substituted by valine (TV226), proline TTP226) or both Thr 226 and Thr 236 substituted by valine (TV228j236). S. &a/is, S. sanguis and S. pneumoniae IgAl pmteases all cleave the Pm 227-Tht226 bond of the wild-type IgAl hinge. Mutant TV226 was resistant only to the S. sanguis IgAl protea&; whereas both TP228 and TV228/236 were resistant to IgAl proteases from S. or&is and S. sanguis. Conclusions:(1) Each IgAl protease appears to possess different requirements for substrate recognition and cleavage. (2) The IgAl pmtease of S. sanguis seems to require Thr at residue 228 for IgAl cleavage. (3) Conformation surrounding residue 228 appears to confer sensitivity to S. oralis IgAl protease action. (4) Thr at residue 228 dos not appear critical for S. pneumoniae IgAl protease action.
I P.3.06.24 I Fc alpha receptor on T lymphocytes subpopulations N. Petre, A. CBlugHru, E. Kozma, G. [email protected] of Immunology Bucharest, Romania Fc receptors for IgA (FccrR)were described on the main cellular types of the immune system, inclusively on a low proportion of T lymphocytes, that increased in different diseases. In this study, the presence of FcaR was determined on the CD4+ and CD8+ T cell subpopulations using high purified T cells obtained from normal donors, freesh isolated by negative selection with MoAb antiCD14, anti-CD19 and antiCD56, on magnetic beads coated with F(ab’)s fragments of goat anti-mouse IgG. The study was carried out by flow-cytometry, using specific antiCD4, anti-CD8, anti-IgA and anti-CD89 F(ab’)2 fragments of antibodies, conjugated with PE or FITC. The bindlng capacity of human IgA and also the presence of the CD89 determinant, was revealed on a low number of both, CD4+ and CD8+ T lymphocytes. The proportion of FcaR carrying cells registered an individual variation ranging between 1.8-2.6 and 2.2-2.7, on the CD4+ and respectively, CD8+ T lymphocytes. A biologic effect consecutive to the interaction ligand-receptor, was also studied by determining the Ca*+ release in the T cell CD4+ and CD8+ subpopulations expressing the FcuR. The correlation between the proportion of FcuR expressing T cell subpopulations and that of Ca*+ releasing cells, account for the role of these receptors in mediating the cellular response.
1P.3.06.25 ] Apparent monovalency of lgG4 Is due to bispeciflcity Janine Schuurman ‘, Ronald van Ree I, Gerrard Perdok ’ , H. Rogier van Doom ‘, K. Yong Tan *, Rob C. Aalberse ‘. ’ Central Laboratory of the Red Cross Blood Tmnsfusion Service and Laboratory of Experimental and Clinical Immunolm Amsterdam, The Netherlands, ’ Schiefand Hospital, Schfedam, The Nethenands
Introduction: Bivalency is an important feature of most IgG antibodies, because it is essential for the formation of biologically effective immune complexes.
Functions
were further anatysed by restriction mapping and sequencing. Expression levels were measuredby FACS analysis of cDNAs transfected in L293 fibroblasts. Reaultsz In the present study, a total of nine FcyRIII-A cDNA clones obtained from two independent NK and LAK cell libraries were analysed. Different types of FcyRIII-A cDNAs were recognized representing four distinct groups of transcripts (al, a2,a4, AEC2) which either contained unique 5-UT sequences due to alternative promoter usage (al, a2, a4), or diverging sequences after the EC1 exon (a4), or were generated by alternative splicing (AECP). The al and a2 transcripts appeared to encode the normal FcyRIII-A receptor. Transfection experiments showed that surface expression of al, a2,and AEC2 is dependent on the FcR y subunit. Fcy FtIII-Aa4/FcR y subunit transfectants were incapable to express any functional Fey Rlll variant on 293 fibroblasts. Different patterns of anti-FcyRIII mAb reactivities were observed for al and a2 compared to AEC2 allowing for the identification of attemative spliced FcyRIII-A gene products on Fey RIII-expressingcells. Con&don: These date indicate. that in addition to the gene duplication event generating the two FcyRIII-A and FcyRIII-B receptors alternative splicing serve to control for the expression of modiiied Fey Rlll receptors. Further studies on the function and tissue distribution are necessary to elucidate the biological significance of such modified Fc receptors.
P.3.06.17
Noo;;;:
as promoter of metallolectin oxidative
A.J. Kulberg, J.B. Berkun. N.F: Gamaieya Epidemiology and Microbiology Institute, Moscow, Russia Introduction: Metalloproteins, like for example ConA, may exist in nonoxidized form if only transition metal(s) is located In nonpolar space of the protein molecule. It suggests that the conformational transitions of the metallolectin molecule resulting hydratation of the metallocanying space would be accompanied by the protein oxidatlve damage(s). To induce such a process we decided to use for ConA treatment the preparation of serum IgG because of amphypatic characteristics for several portions of its molecule (A.J. Kulberg et al., 1960-l 996). Msterlals and Mahods: Commercial preparations of ConA, human IgG and human serum albumin (control) were mixed in different proporttons followed by testing hemaggluttnation activity of ConA against human erythrocytes, U.V. analysis of the preparations tested and their molecular characteristics by ultrafiltration technique. Resulte: Adding IgG in over increasing concentrations to ConA results in rising inhibition of the ConA hemagglutination activity. The effect of serum albumin upon the ConA activity was negligible. Equimolar mixture of ConA and IgG versus ConA and IgG tested separately shows decrease in U.V. absorption, predominantly between 210-240 nm. The phenomenon of hypochromy doesn’t accompany any cleavage of the ConA-IgG mixture. However, when the above mixture is irradiated additionally wlthin minutes at 260 nm a clearly detected cleavage of the proteins (predominantly ConA) doss occur. Conclusion: Interaction ConA with IgG provokes change in ConA conformation revealing in U.V. spectral characteristics and loss of hemagglutination activity. The state of the complex formed can be considered as an intermediate for the further protooxidative cleavage of metallolectin and IgG. It may imply that the biological activity of lectins is realized through activation of their oxidative potency mediated by transition metal(s) co-ordinated by these proteins.
1 P.3.06.16
25 June 1997 - Poster presentations
of Zg isotypes ana’ their receptors
1 IgG-mediated anaphylaxis and inflammation are Impaired in mice lacking FcyRlll
W.L.W. Hazenbos’, J.E. Gessner*, F.M.A. Hofhuis’, H. Kuipers’, D. Meyers, I.A.F.M. Heijnen’, R.E. Schmidt2, M. Sandor3, P.J.A. CapeI’, M. Da&on4, J.G.J. van de Winkel ‘, J.S. Verbeek’. ’ Dept. oftmmunology, University Hospital lltrecht, The Nethettands, 2Dept. of Clinical Immunolo~ Me&al School, Hannover, Germany 3fatho/ogy and Lab. Medicine, University of Wisconsin, USA, 4Lab. d’lmmunologie Cellulaire et Clinique, Inst. Curie, Paris Extensive in vitro studies have demonstrated that various IgG-mediated effector functions of the immune system are triggered by interaction between antigenbound IgG and receptors for IgG (FcyR). In order to study the contribution of FcyR to in vivo immune reactions, we have generated mice deficient in the ligand binding IYchain of FcyRIII. Macrophages from these mice failed to bind and ingest particles opsonized with IgGi, in contrast to IgG2a or IgGPb. indicating an apparent specificity of IgGl for FcyRIII, the in vivo relevance of which is currently under further investigation. IgGtriggered in vitro degranulation of mast cells, and in vivo passive cutaneous anaphytaxis were abrogated in FcyRIII-‘- mice, while both reactions developed normally after triggering with IgE. In addition, after being depleted of complement, FcyRIII-‘- mice did not elidt an Arthus reaction, the classical model for IgG-mediated inflammation. In sum, FcyRlll is essential for recognition of complexed IgGl and for IgG-mediated anaphylaxis and complement-independent inflammation. These findings indicate a therapeutic potential for interfering in the function of FeyRIII.
P.3.06.19
FcyRll slgnal transduction in neutrophlls is differently regulated by receptor-type protein-tyroslne phosphatases CD45 and CD148 (HPTPr$DEP-1)
M. Hundt, R.E. Schmidt. Department of Clinical Immunology Hannover Medical School, Germany Introduction: Activation of the 40 kDa low-affinity receptor for IgG (FcyRII, CD32) leads to tyrosine phosphorylation, mobilisation of intracellular calcium and production of superoxide anions in neutrophils. It has been established that protein-tyrosine kinases and phosphatases am essential for the regulation of Intracellular signatllng. CD45 is a type I receptor-type protein-tyrosine phosphatase (RPTP) with two enzymatically catalytic regions. Recently it has been demonstrated that co-cross-linking of CD45 modulates the signal transduction pathway of FcyRll in neutrophils. In contrast, the functional characteristics of CD146, a type Ill RPTP wtth only one phosphatase domain, is unknown. Mstsrlals and Mahods: The expression of CD45 and CD146 was analysed by indirect immunoftuorescence with saturating amounts of mouse monoclonal antibodies (mAb) of the IgGI isotype: ICO-166 (isotype controt), 7E.12 (CD45), and 143-41 (CD146). The rise of free intracellular calcium was measured by flow cytometry using fluo-3. Neutrophils were preincubatad with as&es of mAb at a dilution of 11100 and than washed with PBS. Subsequently FcyRll was cross-linked by mAb IV.3 (IgGsb, ascites, l/600) and F(ab’)s goat anti-mouse (200 &ml). The production of superoxide anions was measured by lucigenin enhanced chemiluminescence after idenfcal pretreatment as described above. Rssulta: Neutrophils express CD46 in higher density than CD146. Co-crosslinking of CD45 with FeyRll reduced calcium mobilisatlon and respiratory burst significantly in comparison to control ascites. In wntrast, prelncubatlon with CD146 decreased superoxide anion generation, but did not change calcium rise: Meanf SEM,N GAMonly ICO-16s
Expression Calcium mobilisation
MFI, 3 MCF, 6
7E.12
(isotvpe contron
(CD4w
14341 (CDW)
.I. 29.2f1.4
7.0zt3.2 54.4fl.7
169.3f65.4 29.9f2.4
52.0fll.3 59.7f3.4
115f66
316f230
146f93
152f69
Superoxide anion generation
counts(x103),3
Conclusion: Bothclustered human RPTP, CD45 and CD146, inhibit FcyRlI induced superoxide anion generation, but they diier in regulation of calcium mobilisation. In previous studies it has been demonstrated that Fey RII induced Hz4 production is only in part dependent on calcium as a second messenger. Therefore, it is suggested that co-cross-linking of FcyRll and CD45 inhibits superoxide anion generation first by calcium independent pathways, second upstream, and possibly third downstream of calcium release in calcium dependent pathways. In contrast, CD146 has to dephosphorylate proteins downstream, but not upstream of calcium mobilisation. These distinct functional capacities may be important for differential regulation by currently unknown ligands of CD45 and CD146 (This work was supported by the Deutsche Forschungsgesellschaft Hu 704/1-l and Sonderforschungsbereich 244/AO9.)
P.3.06.20
Molecular analysis of the function of the human IgA Fc domain
J.M. Hexham, L.N. Carayannopoulos, J.D. Capm. Mo/ecu/ar lmmunofogy Center, Dept MicrobioiogK UT Southwestern Medical Center at Dallas 6000 Harry Hines Blved, Da//as, TX, USA Introduction: IgA is the immunoglobulin subclass largely responsible for humoral immune protection at the exposed mucosal surface. However relatively liile is known about the effector functions of the IgA Fc region. We have used a panel of mutants to define the sites on IgA for the specific IgA Fc receptor binding and the polymeric immunoglobulin receptor binding. Materials and Methods: IgA/lgG domain swap mutants as well as point and truncation mutants of human IgAl were constructed and expressed as recombinant baculoviruses. Binding of these mutant IgA molecules was assayed by rosetting with the IgA specific Fc receptor expressed on the HL60 cell line. Binding to the polymeric immunoglobulin receptor was measured by FACS of the receptor-expressing cell line MDCK and by ELISA using baculovirus expressed poly Ig receptor. Resutk The IgA specific Fc receptor binds to IgA at the junction of the CH2 and CH3 domains, in a position analogous to the site used by Staphlococcal Protein A in its interaction with IgG. The poly lg receptor binds to dime& IgA in the CH3 domain. Binding is specific to dimetic IgA. The role of tailpiece and J chain, which are wmponents of the complex is presently under investigation. We are also attempting to minimise the binding domain to enable efficient targetting of lgkbased constructs to the mucosla surface.
Functions of Ig isotypes and their receptors
25 June 1997 - Poster presentations
Conclueions: The disection of the molecular functions of IgA and other antibodies will enable the construction of antibodies with enhanced or modffed effector functions. In particular the poly lg receptor binding motif may be used to target antibodies or drugs to mucous membranes.
( P.3.06.21
1 Clinical value of soluble IgG Fc receptor type III (sFcyRIII) in plasma from patients with neutropenia
Harry R. Koene ‘, Masja de Haas ’ , Marion Kleijer ‘, Tom W.J. Huizinga *, Dirk Roes’, Albert E.G.Kr. von dem Borne ‘a3.’ Central Labomtory of the Netherfands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunolosv, Univemity of Amsterdam, Amsterdam, The Nethetlands, * Unfversfty Hospital Leiden, Department of Rheumatof~, Amsterdam, The Netherlands, 3Academic Medical Centre, Department of Hematol~, Amsterdam, The Netherlands Introductfon: Previous studies have shown that the plasma level of soluble IgG Fc receptor type Ill (sFcyRIII) is a measure of the total body neutrophil mass. The aim of this study was to determine whether the plasma level sFcy Rlll can bs used as a parameter to assess the risk of contracting bacterial infections for patients with neutropenia. Methods: We measured sFcyRlll levels in plasma of patients with neutropenla of unknown origin, whose blood was sent to our laboratory for diagnostic evaluation of neutropenia (neutrophil count < 1500@). Clinical data from 66 neutmpenic patients were obtained from the patient files. The occurrence or absence of bactedal infections was scored in the period of three months before to three months after a single sFcy Rlll measurement. In addition, longitudinal data were obtained from 21 patients. Reeultr: of the 66 included neutropenic patients, 15 had suffered from a bacterial infection. Neither the age distribution, nor the genotype frequencies of neutmphil FcyR polymorphisms were different among the group with and without infections. Both neutmphil count and plasma level sFcy Rlll were significantly lower in the patient group with infections, compared to the non-infected group (p = 0.03 and p < 0.0001, respectively). No infections were reported for patients who had plasma sFcyRlll levels above 100 arbiirary units (AU; N: 40-180). After matching each infected patient with two non-infected patients having the same neutrophil count, sFcy Rlll plasma levels remained significantly lower in the group with infections (p = 0.0001). For the patients who were followed in time (mean follow-up: 9 months), no infections were reported when sFcyRlll levels were above 100 AU. Conotuelon: A plasma sFcyRlll level above 100 AU in patients with neutmpenia might indicate a normal bone marmw neutmphil production and therefore no increased risk of contracting bacterial infections.
1P.3.06.22 1 Discovery of an adenosine deaminase activity in the preparates of human IgO3 Andrey V. Zavialov, V.P. Zav’yalov. lnstftute of /mmuno/ogica/ Engineering, 142380 Lyubuchany, Chekhov District, Moscow Region, Russia Introduction: Earlier by means of radio ligand binding assay we have shown that the immobilised IgG and Bence-Jones proteins bind pudne nucleotide monophosphates and nucleosides with high specific affinity (G = 2 x 1O-g M for cGMP and inosine. Kd = 8 x lo-’ M for CAMP, & = 6 x lo-’ M for AMP and GMP). Materialsand Methods: Human IgG was separated from the blood plasma of heaithy donors by the ion exchange chromatography. The lgG3 subclass was separated from other subclasses on the Sephamse Protein A column, and continuously treated by ion exchange chromatography on MonoC and gel filtration on Supemse 12 columns. Reeults: We discovered that the fractions of IgG from the blood plasma of healthy donors eluted from the ion exchange column possess an adenosine deaminase activity. More than 90% of the activity is related to the lgG3 subclass separated from other IgG subclasses on the Sephamse Protein A column and continuousiy treated by ion exchange chromatography on MonoC and gel filtration on Supemse 12 columns. The adenosine deaminase activity of lgG3 is characterfsed by Michaelis constants Ku = 2 + 3 x 1O-3 M and Km = 20 t 30 k-l. Involving activity disappears after modification of tryptophan residues or after reduction and alkylation of disulphide bonds in non-denatured conditions. It was found that the lgG3 inriches 20-fold of an adenosine deaminase activity after gel filtration in 8M urea and renaturation. Conclusion: The data described above permit to suggest that the hinge region of lgG3 provides access to the adenosine deaminase active site(s) located in the constant parts of Fab subunit.
P.3.06.23
403
Elucidation of cleavage site requirements for human IgAl hinge cleavage by etreptococcal IgAl PrOteaseS
MR. Batten, J.M. Woof. Department of Mofecufar and Cellular Pathology, University of Dundee Medical School, Ninewells Hospftal, Dundee, UK
Introduction:IgA is the major immunoglobulin isotype in mucosal secretions, with its main role being defence of the mucosae against bacterial and viral invasion. S-IgA is resistant to most pmteolytic degradation, with the exception of the IgAl proteases, produced by pathogenic bacteria responsible for diseases such as pneumonia, meningitis and periodontal disease. Cleavage of IgAl into Fab and Fc fragments results in loss of structural integdty and antibacterial function, facilitating mucosal colonisation and establishment of infection. Cleavage occurs at specific bonds within the IgAl hinge region, although the exact requirements for substrate recognition and cleavage have not been elucidated. This study aims to determine the precise structural requirements for IgAl cleavage by the IgAl pmteases. Materielsand Methods:Site-directed mutagenesis by PCR overlap extension was performed on a plasmid based on the mammalian expression vector, pEE6HCMV. The vector contains the IgAl heavy chain gene, permitting manipulation of the hinge region. Constructs were expressed in an established CHO cell expression system to produce chimeric anti-NIP human IgAl antibodies. Each antibody was incubated with log-phase bacterial cufture from three streptococcal strains, then immunoblotted with anti-IgA antibodies to assess cleavage susceptibility. Resutts: Three mutants were analysed in the study. These have Thr 226 substituted by valine (TV226), proline TTP226) or both Thr 226 and Thr 236 substituted by valine (TV228j236). S. &a/is, S. sanguis and S. pneumoniae IgAl pmteases all cleave the Pm 227-Tht226 bond of the wild-type IgAl hinge. Mutant TV226 was resistant only to the S. sanguis IgAl protea&; whereas both TP228 and TV228/236 were resistant to IgAl proteases from S. or&is and S. sanguis. Conclusions:(1) Each IgAl protease appears to possess different requirements for substrate recognition and cleavage. (2) The IgAl pmtease of S. sanguis seems to require Thr at residue 228 for IgAl cleavage. (3) Conformation surrounding residue 228 appears to confer sensitivity to S. oralis IgAl protease action. (4) Thr at residue 228 dos not appear critical for S. pneumoniae IgAl protease action.
I P.3.06.24 I Fc alpha receptor on T lymphocytes subpopulations N. Petre, A. CBlugHru, E. Kozma, G. [email protected] of Immunology Bucharest, Romania Fc receptors for IgA (FccrR)were described on the main cellular types of the immune system, inclusively on a low proportion of T lymphocytes, that increased in different diseases. In this study, the presence of FcaR was determined on the CD4+ and CD8+ T cell subpopulations using high purified T cells obtained from normal donors, freesh isolated by negative selection with MoAb antiCD14, anti-CD19 and antiCD56, on magnetic beads coated with F(ab’)s fragments of goat anti-mouse IgG. The study was carried out by flow-cytometry, using specific antiCD4, anti-CD8, anti-IgA and anti-CD89 F(ab’)2 fragments of antibodies, conjugated with PE or FITC. The bindlng capacity of human IgA and also the presence of the CD89 determinant, was revealed on a low number of both, CD4+ and CD8+ T lymphocytes. The proportion of FcaR carrying cells registered an individual variation ranging between 1.8-2.6 and 2.2-2.7, on the CD4+ and respectively, CD8+ T lymphocytes. A biologic effect consecutive to the interaction ligand-receptor, was also studied by determining the Ca*+ release in the T cell CD4+ and CD8+ subpopulations expressing the FcuR. The correlation between the proportion of FcuR expressing T cell subpopulations and that of Ca*+ releasing cells, account for the role of these receptors in mediating the cellular response.
1P.3.06.25 ] Apparent monovalency of lgG4 Is due to bispeciflcity Janine Schuurman ‘, Ronald van Ree I, Gerrard Perdok ’ , H. Rogier van Doom ‘, K. Yong Tan *, Rob C. Aalberse ‘. ’ Central Laboratory of the Red Cross Blood Tmnsfusion Service and Laboratory of Experimental and Clinical Immunolm Amsterdam, The Netherlands, ’ Schiefand Hospital, Schfedam, The Nethenands
Introduction: Bivalency is an important feature of most IgG antibodies, because it is essential for the formation of biologically effective immune complexes.