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Mechanism of activation of the classical complement pathway in human serum by a lectin from Euonymus europeus
ABSTRACTS
1401
HUMAN BlH GLOBULIN AND C3b STIMULATE THE RESPIRATORY BURST IN HUMAN MONOCYTES. R.E. Schopf, K.P. Hammann, 0. Scheiner, E.-M. Lemmel, and M.P. Dierich. Institute for Medical Microbiology and Immunolgy, Johannes Gutenberg University, D-6500 Mainz, West Germany. BlH globulin (flH) is a regulator protein in serum of the alternative pathway of complement activation. Based on recent findings, pointing toward the presence of @lH binding sites on lymphoid and phagocytic cells, we asked whether human monocytes (Me) could be affected by81 H. Activation of Me by appropriate stimuli results in a series of metabolic events terms “respiratory burst” which can be measured by nitro blue tetrazolium (NBT) reduction or by chemiluminescence (CL). Human Me from the peripheral blood were incubated with highly purified@lH and for comparison with human C3b and with zymosan particles. In CL as well as in NBT reduction the stimulatory effect of BlH was time- and dose-dependent. In CL the peak of stimulation by131H was found after about 15 minutes of incubation with 28 ugPiH/ml. No further increase in Me activation could be obtained by increasing theBlH concentration up to 83 uglml. C3b applied at 19 to 167 uglml and zymosan particles (330 uglml) were also found to be highly active, but the maximum was reached here after 45 minutes. With the highest amount of C3b no plateau was reached, yet. In NBT reduction, C3b rather inhibited Me function at the concentrations of 16,31, and 63 uglml. The differences between NBT reduction and CL could possibly be attributed to the measurement of only cell-bound reductive potentials by NBT reduction, while in CL measurements also products of the extracellular space are assessed. MB depletion and enrichment studies point at Me as being responsible for the stimulatory effects observed. Our results suggest thatPlH and C3b are potent stimulators of human Me. Supported by Deutsche Forschungsgemeinschaft Scho 273/1-l; and DFGlSFB 101, A5
THE ROLE OF C3 FRAGMENTS IN POLYMORPHONUCLEAR LEUKOCYTE (PMN) MEDIATED CYTOCIDAL REACTIONS. RD. Schreiber’, A.B. Bjornson and M.K. Pangburn. Res. Inst. of Scripps Clinic, La Jolla, Ca. and Univ. of Cincinnati, OH. Human PMN display the ability to bind particles coated with C3b or CBbi in absence of immunoglobulin (lg). Using purified fragments of C3 we have confirmed that these cells possess two distinct C3 receptors which display specificity for their respective ligands but not for C3d. PMN rosettes with erythrocytes (E) carrying only C3b (EC3b) were inhibited by soluble C3b but not CBbi while EC3bi rosettes were inhibited by soluble C3bi but not C3b. Neither rosetting reaction was inhibited by native C3. C3 which was modified at the thioester site by treatment with methylamine or repeated cycles of freezing and thawing was comparable to C3b in inhibitory activity. Engagement of either C3 receptor elicited comparable biological responses from PMN. Particle bound C3b or CBbi stimulated the production of active oxygen species by PMN as detected by chemiluminescence (CL). CL responses occurred in total absence of lg and were inhibited by sodium azide, superoxide dismutase or cytochalasin B but not by catalase. Using a %r release assay, purified PMN lysed tumor cells (Raji) or E coated only with antibody (Ab) but did not lyse cells carrying only C3 fragments. However, in presence of limiting amounts of Ab, cell bound C3 fragments enhanced PMN cytocidal activity lo-25 fold. Ab was not required to effect phagocytosis of C3 fragment coated E. coli 04. Incubation of purified PMN and FITC-labelled E. coli with a mixture of the 6 alternative pathyway proteins at their respective physiological concentrations led to ingestion of the bacteria as assessed by a combination of light, fluorescent and electron microscopic techniques. Heat inactivation of the protein mixture abrogated the phagocytic response. Thus these results illustrate the participation of particle bound C3 fragments in promoting both antibody dependent and independent cytotoxic reactions mediated by PMN.
MECHANISM OF ACTIVATION OF THE CLASSICAL COMPLEMENT PATHWAY IN HUMAN SERUM BY A LECTIN FROM Euonymus europeus. D.R. Schultz’ and P.I. Arnold, Dept. of Medicine, University of Miami School of Medicine, Miami, Florida. A lectin from Euonymus europeus (EE) is most specific for the blood group B determinant (J. Petryniak et al, Arch. Biochem. Biophy. 178:118, 1977). When purified EE was added to normal human serum (NHS) at 37°C for 30 min (21 uglml), Cl, C4 and C2 were consumed but not C3. Amounts greater th_an 21 uglml caused C3 consumption as well. Since EE had no effect on purified C4, C2, C3, or on the Cl inhibitor in serum, we studied its mechanism of action. EE caused activation of the classical C pathway after it was treated with pronase or boiled, but lost its activity after treatment with periodate. This indicated that the carbohydrate portion of EE may be involved in C activaticn. 1251-EE did not bind to fluid phase or cellbound purified human Clq or to purified precursor Cl or Cl, but it did cause the conversion of Cl-Cl in NHS at 37°C. EE did not activate Cl in a hypogammaglobulinemic serum, but upon reconstitution of the serum with heat-inactivated (56°C 30 min) normal r-globulin, C4 and C2 were consumed. A monoclonal antibody (vM) to blood group B determinant aIS0 caused the consumption of C4 and C2 after addition to NHS at 37%. Synthetic blood group B determinant linked to a carrier [aD gal(l-3)aL fuc (1-2) B D gal-0-8-methoxyocty! carbonyl glycoside] partially inhibited the consumption of C4 and C2 by the lectin in NHS. We conclue that both EE and the monoclonal antibody to blood group B determinant bind to a substance similar to or the same as blood group B substance in NHS to cause activation of the classical C pathway. They-globulin may be a third factor in addition to EE and determinant B for activation of the classical C pathway in this reaction. Supported by the Kroc Foundation and by Chembiomed Ltd, Edmonton, Alberta, Canada.
1401
HUMAN BlH GLOBULIN AND C3b STIMULATE THE RESPIRATORY BURST IN HUMAN MONOCYTES. R.E. Schopf, K.P. Hammann, 0. Scheiner, E.-M. Lemmel, and M.P. Dierich. Institute for Medical Microbiology and Immunolgy, Johannes Gutenberg University, D-6500 Mainz, West Germany. BlH globulin (flH) is a regulator protein in serum of the alternative pathway of complement activation. Based on recent findings, pointing toward the presence of @lH binding sites on lymphoid and phagocytic cells, we asked whether human monocytes (Me) could be affected by81 H. Activation of Me by appropriate stimuli results in a series of metabolic events terms “respiratory burst” which can be measured by nitro blue tetrazolium (NBT) reduction or by chemiluminescence (CL). Human Me from the peripheral blood were incubated with highly purified@lH and for comparison with human C3b and with zymosan particles. In CL as well as in NBT reduction the stimulatory effect of BlH was time- and dose-dependent. In CL the peak of stimulation by131H was found after about 15 minutes of incubation with 28 ugPiH/ml. No further increase in Me activation could be obtained by increasing theBlH concentration up to 83 uglml. C3b applied at 19 to 167 uglml and zymosan particles (330 uglml) were also found to be highly active, but the maximum was reached here after 45 minutes. With the highest amount of C3b no plateau was reached, yet. In NBT reduction, C3b rather inhibited Me function at the concentrations of 16,31, and 63 uglml. The differences between NBT reduction and CL could possibly be attributed to the measurement of only cell-bound reductive potentials by NBT reduction, while in CL measurements also products of the extracellular space are assessed. MB depletion and enrichment studies point at Me as being responsible for the stimulatory effects observed. Our results suggest thatPlH and C3b are potent stimulators of human Me. Supported by Deutsche Forschungsgemeinschaft Scho 273/1-l; and DFGlSFB 101, A5
THE ROLE OF C3 FRAGMENTS IN POLYMORPHONUCLEAR LEUKOCYTE (PMN) MEDIATED CYTOCIDAL REACTIONS. RD. Schreiber’, A.B. Bjornson and M.K. Pangburn. Res. Inst. of Scripps Clinic, La Jolla, Ca. and Univ. of Cincinnati, OH. Human PMN display the ability to bind particles coated with C3b or CBbi in absence of immunoglobulin (lg). Using purified fragments of C3 we have confirmed that these cells possess two distinct C3 receptors which display specificity for their respective ligands but not for C3d. PMN rosettes with erythrocytes (E) carrying only C3b (EC3b) were inhibited by soluble C3b but not CBbi while EC3bi rosettes were inhibited by soluble C3bi but not C3b. Neither rosetting reaction was inhibited by native C3. C3 which was modified at the thioester site by treatment with methylamine or repeated cycles of freezing and thawing was comparable to C3b in inhibitory activity. Engagement of either C3 receptor elicited comparable biological responses from PMN. Particle bound C3b or CBbi stimulated the production of active oxygen species by PMN as detected by chemiluminescence (CL). CL responses occurred in total absence of lg and were inhibited by sodium azide, superoxide dismutase or cytochalasin B but not by catalase. Using a %r release assay, purified PMN lysed tumor cells (Raji) or E coated only with antibody (Ab) but did not lyse cells carrying only C3 fragments. However, in presence of limiting amounts of Ab, cell bound C3 fragments enhanced PMN cytocidal activity lo-25 fold. Ab was not required to effect phagocytosis of C3 fragment coated E. coli 04. Incubation of purified PMN and FITC-labelled E. coli with a mixture of the 6 alternative pathyway proteins at their respective physiological concentrations led to ingestion of the bacteria as assessed by a combination of light, fluorescent and electron microscopic techniques. Heat inactivation of the protein mixture abrogated the phagocytic response. Thus these results illustrate the participation of particle bound C3 fragments in promoting both antibody dependent and independent cytotoxic reactions mediated by PMN.
MECHANISM OF ACTIVATION OF THE CLASSICAL COMPLEMENT PATHWAY IN HUMAN SERUM BY A LECTIN FROM Euonymus europeus. D.R. Schultz’ and P.I. Arnold, Dept. of Medicine, University of Miami School of Medicine, Miami, Florida. A lectin from Euonymus europeus (EE) is most specific for the blood group B determinant (J. Petryniak et al, Arch. Biochem. Biophy. 178:118, 1977). When purified EE was added to normal human serum (NHS) at 37°C for 30 min (21 uglml), Cl, C4 and C2 were consumed but not C3. Amounts greater th_an 21 uglml caused C3 consumption as well. Since EE had no effect on purified C4, C2, C3, or on the Cl inhibitor in serum, we studied its mechanism of action. EE caused activation of the classical C pathway after it was treated with pronase or boiled, but lost its activity after treatment with periodate. This indicated that the carbohydrate portion of EE may be involved in C activaticn. 1251-EE did not bind to fluid phase or cellbound purified human Clq or to purified precursor Cl or Cl, but it did cause the conversion of Cl-Cl in NHS at 37°C. EE did not activate Cl in a hypogammaglobulinemic serum, but upon reconstitution of the serum with heat-inactivated (56°C 30 min) normal r-globulin, C4 and C2 were consumed. A monoclonal antibody (vM) to blood group B determinant aIS0 caused the consumption of C4 and C2 after addition to NHS at 37%. Synthetic blood group B determinant linked to a carrier [aD gal(l-3)aL fuc (1-2) B D gal-0-8-methoxyocty! carbonyl glycoside] partially inhibited the consumption of C4 and C2 by the lectin in NHS. We conclue that both EE and the monoclonal antibody to blood group B determinant bind to a substance similar to or the same as blood group B substance in NHS to cause activation of the classical C pathway. They-globulin may be a third factor in addition to EE and determinant B for activation of the classical C pathway in this reaction. Supported by the Kroc Foundation and by Chembiomed Ltd, Edmonton, Alberta, Canada.