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Interactions between MBL and the MASP proteins in lectin pathway hemolysis
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252-8 REGULATION OF THE INFLAMMATORY RESPONSE TO Ne/eeeria mroingitidja Bl940 BY MANNOSE-BINDING LECTIN DL Jackl.3, U Vogel?, AJ Ten&, M Frosch? MW Turner’, NJ Klein’ ‘Inst. of Child Health, London, United Kingdom; 2Univ. California, Irvine, CA Mannose-binding lectin (MBL) is a serum collectin which binds to the repeating sugar residues commonly found on micro-organisms. Genetic MBL deficiency has been linked to an increased susceptibility to meningococcal disease. We examined the interacfion of MBL with isogenic mutants of serogroup B meningccccci which differed in the expression of capsular polysaccharide and the LPS sialic acid acceptor site. Bacteria preincubated with 2pg/ml purified MBL were mixed with dextran separated leukocytes in serum-free media. MBL increased the phagocytosis of most mutants by neubophils at 60 min but this was not simply related to Ihe amount of MBL bound. In the same assay MBL delayed the loss of CD621 and the increase of CDtlb by neutrophils at 39 min indicating a delay in cellular activation. This effect was also observed in MBLdeficient anticoagulated blood that had been supplemented with MBL, but this required more MBL (8 pglml) and was observed at an earlier time point (15 min). We investigated the effect of MBL on monocyts cyiokine responses using an intracellular staining technique. Brefeldin A and diiring wncentrationz of MBL were added to whole blood from MBL defident indiiuds. We observed a slight increase in TNFa at low concentrattons and a deaease at high concentrations. The effect was similar buf more pronounced witi IL6 whereas ILlp production was enhanced over a much wider range of MBL concentrattons. These results indicate that MBL exerts a powerful but complex modulation over the inflammatory response to N. menhgtidis This may explain our unusual phagocytosis results and some of the innate variation in dinical responses to meningococcal disease.
253- MENINGOCOCCAL LIPOPOLYSACCHARIDE StALYLATlON ALTERS THE BACTERICIDAL ACTMTY OF HUMAN MANNOSEBINDING LECTIN DL Jackl.3, GA Jar&*, CL Booth’, J Liz, NJ Klein’ and MWTumerl ‘inst. of Child Health, London, United Kingdom: 2Univ. of CA and VA Med. Cntr., Ban Francisco, CA; JDivision of Molecular and Genetic Medicine, Sheffield, United Kingdom Mannosebinding lecttn (MBL) is a serum collectin which binds to the repeating sugar residues commonly found on micm-organisms and activates the complement system in an antibody-independent manner. Genetic MBL deficiency has teen linked to an increased susceptibility to meningoccccal disease. We examined the interaction of MBL with three dinical isolates of serogmup C meningococci which differed in endogenour LPS sialylation and serum sensitivity. MBL and monoclonal antibody 3Fll (which recognises non-sialyiated LPS) were shown to bind simultaneously to strain 8026 (4% endogenous sialylatton) by dual colour flow cyiomeby Binding of both these reagents was greatly reduced when the organisms were grown in the presence of exogenous CMP-NANA to increase LPS sialylation. The binding of MBL or 3Fll to strains 7954 and 7973 (48% ant 86% endogenous sialylation, respectively) was below the detection limit of flow cytometry Preincubation of stin 8028 with MBL increased kilting by 50% using 0.5% MBLdeficient serum and this coincided with a slight increase in C4 binding. In 10% MBL deficient serum there was a clear increase in C4 and C5b-9 binding to the non-sialylated form with the addition of exogenous MBL. Using the more sensitive technique of transmission elecfmn microscopy with immunogold detectkm, we could still detect MB1 binding to sialylated 8026 although this was greatly rw compared to the non-sidylated form and appeared to be limited to outer membrane blebs. We mndude that MBL can bind to encapsulated serogroup C meningococci to inaease complement killing.
254-t STUDY OF MBLMASPS INTERACTION INHIBITOR AND c$-MACROGLOBULIN M Kojima and RB Sim. MRC Immunochemistry Unit, Department University of Oxford, Oxford OX1 3QU, UK.
of
WITH
Cl
Biochemistry,
Serum MBL is a collectin. which binds to carbohydrates, such as mannan, and activates the &tin pathway of complement via MASPI and MASP2. Although the most recently discovered, the pathway is thought to predate the others, hence the regulation of its fust component, the MBL-MASPs complex, is of much Interest. In this study, interaction of MBL-MASPs bound to a rnannancoated surface with reported inhibitors of MASPs was investigated. Purltied MBL-MASR was radiolabelled with ‘4, bound to manuan-coated microtitre plates and incubated with Cl-inh and/or a,M. Approximately 10% of of the mannan-bound radIoactivity was released into the supematant on incubation (1 h, 37’C) with buffer or with a serpin control, ovalbumm (0.2 mg/ml). A similar amount of radioactivity was released when the wells were incubated with %M, hence no significant interaction between lzsIMBL-MASPs and %M was revealed in this experiment, consistent with other work in this laboratory. When ‘zI-MBL-MASPs bound to a mannan-coated surface was Incubated with Cl-i&. there was release of radioactivity into the fluid phase, which correlated with Cl-inh concentration. Analysis of species in the supernant revealed that Cl-inh-MASP complexes were among the components released. Cl-inh is the only physiological regulator of Cl complex: it forms covalent complexes with activated Clr and Cls and dissociates them from Clq. Currently, it is not established whether Cl-inh causes selective dissociation of the MASPs from MBL or it mediates dissociation of the whole complex from mannan.
255. INTERACTIONS BETWEEN MBL AND THE MASP PROTEINS IN LECTIN PATHWAY HEMOLYSIS. B. Xu’, Y. Zhang’, K. Takahash?, A.R. Ezekowitz’, T.F. Lint’ and H. Gewurz’. Rush Medical College* and Harvard Umversity! MBL was purified from human serum by elution from mannan (M)-substituted Sepharose and additional column chromatography. MBL complexed with MASP-1, activated MASP-2 and MApl9 (MBL-MASP’), as confirmed by SDS-PAGE, Western blots using anti-MASP peptide antisera and C4 bmding, sensitized M-coated E (E-M) for lysis in human and guinea pig serum in Mg-EGTA; guinea pig serum was at least IO-fold more effective. E-M sensitized with rMBL or MBL from serum purified free of associated MASP* lacked this activity. E chemically coated with MBL-MASP* in the absence of M also lysed in serum-Mg-EGTA, but E coated with MBL free of MASP* did not. E-M sensitized and E chemically coated with MBL-MASP* both Iost lysability in serum-Mg-EGTA when treated with serine protease inhibrlors, EDTA or EGTA, even as MBL remained bound to the cell as previously described, indicating calcium-dependence of the MBL-MASP* complex. However, much more EDTA and EGTA were required for removal of MASP* from MBL than for removal of Clrlsz from Clq. E-M-MBL-MASP* could be formed upon incubation of MBL lackmg MASP* with E-M in MBLdepleted serum as a MASP source, with optimal activity observed when MBL reacted with E-M in the presence of the MASPs. This reaction served as a sensitive assay for MASP* activity, with titers 2300 hemolytic unils/ml in normal buman serum. These experiments emphasize the extremely stable MBL-MASP* complex formed when MBL reacts with M in the presence of the MASPs, particularly iu hemolytic activity and the strength with which It binds calcium. and introduce a sensitive assay for MASP* hemolytic activity.
252-8 REGULATION OF THE INFLAMMATORY RESPONSE TO Ne/eeeria mroingitidja Bl940 BY MANNOSE-BINDING LECTIN DL Jackl.3, U Vogel?, AJ Ten&, M Frosch? MW Turner’, NJ Klein’ ‘Inst. of Child Health, London, United Kingdom; 2Univ. California, Irvine, CA Mannose-binding lectin (MBL) is a serum collectin which binds to the repeating sugar residues commonly found on micro-organisms. Genetic MBL deficiency has been linked to an increased susceptibility to meningococcal disease. We examined the interacfion of MBL with isogenic mutants of serogroup B meningccccci which differed in the expression of capsular polysaccharide and the LPS sialic acid acceptor site. Bacteria preincubated with 2pg/ml purified MBL were mixed with dextran separated leukocytes in serum-free media. MBL increased the phagocytosis of most mutants by neubophils at 60 min but this was not simply related to Ihe amount of MBL bound. In the same assay MBL delayed the loss of CD621 and the increase of CDtlb by neutrophils at 39 min indicating a delay in cellular activation. This effect was also observed in MBLdeficient anticoagulated blood that had been supplemented with MBL, but this required more MBL (8 pglml) and was observed at an earlier time point (15 min). We investigated the effect of MBL on monocyts cyiokine responses using an intracellular staining technique. Brefeldin A and diiring wncentrationz of MBL were added to whole blood from MBL defident indiiuds. We observed a slight increase in TNFa at low concentrattons and a deaease at high concentrations. The effect was similar buf more pronounced witi IL6 whereas ILlp production was enhanced over a much wider range of MBL concentrattons. These results indicate that MBL exerts a powerful but complex modulation over the inflammatory response to N. menhgtidis This may explain our unusual phagocytosis results and some of the innate variation in dinical responses to meningococcal disease.
253- MENINGOCOCCAL LIPOPOLYSACCHARIDE StALYLATlON ALTERS THE BACTERICIDAL ACTMTY OF HUMAN MANNOSEBINDING LECTIN DL Jackl.3, GA Jar&*, CL Booth’, J Liz, NJ Klein’ and MWTumerl ‘inst. of Child Health, London, United Kingdom: 2Univ. of CA and VA Med. Cntr., Ban Francisco, CA; JDivision of Molecular and Genetic Medicine, Sheffield, United Kingdom Mannosebinding lecttn (MBL) is a serum collectin which binds to the repeating sugar residues commonly found on micm-organisms and activates the complement system in an antibody-independent manner. Genetic MBL deficiency has teen linked to an increased susceptibility to meningoccccal disease. We examined the interaction of MBL with three dinical isolates of serogmup C meningococci which differed in endogenour LPS sialylation and serum sensitivity. MBL and monoclonal antibody 3Fll (which recognises non-sialyiated LPS) were shown to bind simultaneously to strain 8026 (4% endogenous sialylatton) by dual colour flow cyiomeby Binding of both these reagents was greatly reduced when the organisms were grown in the presence of exogenous CMP-NANA to increase LPS sialylation. The binding of MBL or 3Fll to strains 7954 and 7973 (48% ant 86% endogenous sialylation, respectively) was below the detection limit of flow cytometry Preincubation of stin 8028 with MBL increased kilting by 50% using 0.5% MBLdeficient serum and this coincided with a slight increase in C4 binding. In 10% MBL deficient serum there was a clear increase in C4 and C5b-9 binding to the non-sialylated form with the addition of exogenous MBL. Using the more sensitive technique of transmission elecfmn microscopy with immunogold detectkm, we could still detect MB1 binding to sialylated 8026 although this was greatly rw compared to the non-sidylated form and appeared to be limited to outer membrane blebs. We mndude that MBL can bind to encapsulated serogroup C meningococci to inaease complement killing.
254-t STUDY OF MBLMASPS INTERACTION INHIBITOR AND c$-MACROGLOBULIN M Kojima and RB Sim. MRC Immunochemistry Unit, Department University of Oxford, Oxford OX1 3QU, UK.
of
WITH
Cl
Biochemistry,
Serum MBL is a collectin. which binds to carbohydrates, such as mannan, and activates the &tin pathway of complement via MASPI and MASP2. Although the most recently discovered, the pathway is thought to predate the others, hence the regulation of its fust component, the MBL-MASPs complex, is of much Interest. In this study, interaction of MBL-MASPs bound to a rnannancoated surface with reported inhibitors of MASPs was investigated. Purltied MBL-MASR was radiolabelled with ‘4, bound to manuan-coated microtitre plates and incubated with Cl-inh and/or a,M. Approximately 10% of of the mannan-bound radIoactivity was released into the supematant on incubation (1 h, 37’C) with buffer or with a serpin control, ovalbumm (0.2 mg/ml). A similar amount of radioactivity was released when the wells were incubated with %M, hence no significant interaction between lzsIMBL-MASPs and %M was revealed in this experiment, consistent with other work in this laboratory. When ‘zI-MBL-MASPs bound to a mannan-coated surface was Incubated with Cl-i&. there was release of radioactivity into the fluid phase, which correlated with Cl-inh concentration. Analysis of species in the supernant revealed that Cl-inh-MASP complexes were among the components released. Cl-inh is the only physiological regulator of Cl complex: it forms covalent complexes with activated Clr and Cls and dissociates them from Clq. Currently, it is not established whether Cl-inh causes selective dissociation of the MASPs from MBL or it mediates dissociation of the whole complex from mannan.
255. INTERACTIONS BETWEEN MBL AND THE MASP PROTEINS IN LECTIN PATHWAY HEMOLYSIS. B. Xu’, Y. Zhang’, K. Takahash?, A.R. Ezekowitz’, T.F. Lint’ and H. Gewurz’. Rush Medical College* and Harvard Umversity! MBL was purified from human serum by elution from mannan (M)-substituted Sepharose and additional column chromatography. MBL complexed with MASP-1, activated MASP-2 and MApl9 (MBL-MASP’), as confirmed by SDS-PAGE, Western blots using anti-MASP peptide antisera and C4 bmding, sensitized M-coated E (E-M) for lysis in human and guinea pig serum in Mg-EGTA; guinea pig serum was at least IO-fold more effective. E-M sensitized with rMBL or MBL from serum purified free of associated MASP* lacked this activity. E chemically coated with MBL-MASP* in the absence of M also lysed in serum-Mg-EGTA, but E coated with MBL free of MASP* did not. E-M sensitized and E chemically coated with MBL-MASP* both Iost lysability in serum-Mg-EGTA when treated with serine protease inhibrlors, EDTA or EGTA, even as MBL remained bound to the cell as previously described, indicating calcium-dependence of the MBL-MASP* complex. However, much more EDTA and EGTA were required for removal of MASP* from MBL than for removal of Clrlsz from Clq. E-M-MBL-MASP* could be formed upon incubation of MBL lackmg MASP* with E-M in MBLdepleted serum as a MASP source, with optimal activity observed when MBL reacted with E-M in the presence of the MASPs. This reaction served as a sensitive assay for MASP* activity, with titers 2300 hemolytic unils/ml in normal buman serum. These experiments emphasize the extremely stable MBL-MASP* complex formed when MBL reacts with M in the presence of the MASPs, particularly iu hemolytic activity and the strength with which It binds calcium. and introduce a sensitive assay for MASP* hemolytic activity.