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Complement activation by myelin: Activation of classical pathway by isolated myelin membranes in the absence of antibody
ABSTRACTS
1407
The intramembraneous portion of the C5b-9(m) cylinder is revealed on the fracture E-face as a circular structure elevated approx. 2 nm above the lipid plateau. In unetched fractures it appears as a solid stub; after etching the appearance changes into a ring, confirming the existence of a central waterfilled pore in the molecule. Allowing for the thickness of the replica, the dimensions of this ring were compatible with an inner diameter of approx. IO nm and a wall thickness of approx. 1 nm as previously determined by negative staining. Analysis of complementary replicas revealed that each. cylinder on the E-face corresponded to a sharply punched defect in the lipid monolayer of the fracture P-face. These defects were deepened by etching. Studies on proteoiytically stripped membranes, in which native membrane particles become densely aggregated, showed that the native particles are effectively excluded from the domain of the cylinder. Deep etching revealed the extramembraneous annulus of the molecule. Similarly, circular openings were found on the cytoplasmic surface of the membrane, being likely candidates for the internal apertures of the cylinder. The results support the contention that the C5b-9(m) complex do penetrate the full thickness of the sheep erythrocyte lipid bilayer to create a true pore walled by the hollow cylindrical molecule. MEMBRANOLYSIS BY ISOLATED C9 IN THE ABSENCE OF OTHER COMPLEMENT PROTEINS J. Tschopp’, E.R. Podack and H.J. Mllllef-Eberhard. Research Institute of Scripps Clinic, La Jolla, California 92037. Human C9 without the participation of CXb-8 has potential membranolytic activity. C9 acquires this activity by heat induced polymerization on membranes. Incubation of purified C9 with carboxyfluorescein containing single bilayer vesicles (egg lecithin) for 1 hr at 46°C resulted in marker release. Incubation with monomeric C9 at 20°C had no effect. Marker release correlated with hydrophobic binding of C9 to the vesicle membrane as revealed by flotation through sucrose density gradients. Acquisition of hydrophobic binding sites by C9 was demonstrated by increased binding of the hydorphobic probe 8-aniline-l-napthtalene sulfonic acid during incubation at 46°C. Vesicle lysis was caused by polymerization of C9 to tubules which inserted themselves into the vesicle membrans as shown by electron microscopy. The image of the membrane bound C9 polymer resembled that of the membrane attack complex (MAC). C9 polymerization was accompanied by an increase in&pleated sheet structure and this conformational change was also characteristic for the vesicle bound C9 polymer and for MAC formation from C5b-8 and C9. CQ polymerization also led to fusion of single bilayer vesicles at high lipid to protein ratio and to disassembly of bilayers at high C9 to lipid ratios. The data indicate that isolated C9 can be transformed from a water soluble monomeric protein to a membrane bound amphiphilic polymer. The membranolytic effect of polymerized C9 may be ascribed to the formation of a transmembrane protein channel in addition to physical effects on phospholipid bilayer structure, (Supported by the Swiss National Science Foundation, American Heart Association El No. 79-149 and USPHS Grants Ai 17354, CA 27489 and HL 16411.) COMPLEMENT ACTIVATION BY MYELIN: ACTIVATION OF CLASSICAL PATHWAY BY ISOLATED MYELIN MEMBRANES IN THE ABSENCE OF ANTIBODY. ~F~dmavafhy ~~ngu~~, Carol Koski and Moon Shin. Unversity of Maryland School of Medicine, Department of Pathology and Neurology, Baltimore, MD21201. Many pathological conditions of the central nervous system (CNS) involve damage to and removal of myelin membrane. Extensive phagocytosis of myelin by macrophages and astrocytes and release of myelin constituents into cerebrospinal fluid and into systemic circulation following CNS injury have been well documented. However, very little is known about the initiation of this membrane damage and mechanism of disposal of the damaged tissue. We are interested in the interaction between complement and myelin membranes and its’ possible role in the disposal of damaged myelin in vivo, because activation of complement generates both opsonin(s) and membrane attack complexes. In the present studies, we found that isolated rat or human CNS myelin consumes human complement in a dose dependent manner in the absence of specific antibodies, and it is about ten times more efficient than peripheral myelin. Activation of complement was demonstrated by showing C3 cleavage in the fresh serum incubated with myelin. CNS myelin incubated with C2 deficient (C2D) serum showed no complement consumption, and no factor B conversion was detected in the serum, thus excluding the alternative pathway activation. The involvement of the classical pathway was shown by the Cl transfer assay. Myelin incubated with C2D serum or with purified Cl, and washed, can transfer Cl activity to recipient cells, EAC4. A NEW CONCEPT OF INDUCING IMMUNE CYTOLYSIS: KILLING OF TUMOR CELLS VIA THE ALTERNATIVE PATHWAY INDUCED BY A COVALENT HYBRID OF MONOCLONAL ANTIBODY AND COBRA VENOM FACTOR. Car/-Wilhelm Vogel” and Hans J. MUller-Eberhard. Research Institute of Scripps Clinic, La Jolla, California. The unique specificity of monoclonal antibodies was combined with the cell killing function of the cytolytic alternative pathway of complement. The functionally CBb-like glycoprotein of cobra venom was linked to a murine monoclonal antibody directed to a human melanoma associated antigen by a disulfide bono using a heterobifunctional crosslinking reagent [N-succinimidyl-3-(2-pyridyldithio) propionate]. The covalent monoclonal antibody-cobra venom factor (CVF) complex exhibited specific cytolytic activity in the presence of normal or C4 deficient serum. It induced killing of melanoma cells but not of LG-2 lymphoblastoid cells or P815 mastocytoma cells. The cytolytic action of the monoclonal antibody-CVF complex was selective in that it was limited to the melanoma cells when these were in mixture with one of the other two cell lines. In absence of serum, the complex was noncytotoxic. The antibody or CVF alone had not cytolytic activity with or without serum. It is concluded that CVF covalently linked to melanoma cell bound antibody forms the stable C31C5 convertase with Factors B and D of the alternative pathway, which in turn causes formation of the membrane attack complex and thereby cell death. (Supported by USPHS Grants Al 17354, CA 27489, HL 16411 and DFG Fellowship No. 303/l.)
1407
The intramembraneous portion of the C5b-9(m) cylinder is revealed on the fracture E-face as a circular structure elevated approx. 2 nm above the lipid plateau. In unetched fractures it appears as a solid stub; after etching the appearance changes into a ring, confirming the existence of a central waterfilled pore in the molecule. Allowing for the thickness of the replica, the dimensions of this ring were compatible with an inner diameter of approx. IO nm and a wall thickness of approx. 1 nm as previously determined by negative staining. Analysis of complementary replicas revealed that each. cylinder on the E-face corresponded to a sharply punched defect in the lipid monolayer of the fracture P-face. These defects were deepened by etching. Studies on proteoiytically stripped membranes, in which native membrane particles become densely aggregated, showed that the native particles are effectively excluded from the domain of the cylinder. Deep etching revealed the extramembraneous annulus of the molecule. Similarly, circular openings were found on the cytoplasmic surface of the membrane, being likely candidates for the internal apertures of the cylinder. The results support the contention that the C5b-9(m) complex do penetrate the full thickness of the sheep erythrocyte lipid bilayer to create a true pore walled by the hollow cylindrical molecule. MEMBRANOLYSIS BY ISOLATED C9 IN THE ABSENCE OF OTHER COMPLEMENT PROTEINS J. Tschopp’, E.R. Podack and H.J. Mllllef-Eberhard. Research Institute of Scripps Clinic, La Jolla, California 92037. Human C9 without the participation of CXb-8 has potential membranolytic activity. C9 acquires this activity by heat induced polymerization on membranes. Incubation of purified C9 with carboxyfluorescein containing single bilayer vesicles (egg lecithin) for 1 hr at 46°C resulted in marker release. Incubation with monomeric C9 at 20°C had no effect. Marker release correlated with hydrophobic binding of C9 to the vesicle membrane as revealed by flotation through sucrose density gradients. Acquisition of hydrophobic binding sites by C9 was demonstrated by increased binding of the hydorphobic probe 8-aniline-l-napthtalene sulfonic acid during incubation at 46°C. Vesicle lysis was caused by polymerization of C9 to tubules which inserted themselves into the vesicle membrans as shown by electron microscopy. The image of the membrane bound C9 polymer resembled that of the membrane attack complex (MAC). C9 polymerization was accompanied by an increase in&pleated sheet structure and this conformational change was also characteristic for the vesicle bound C9 polymer and for MAC formation from C5b-8 and C9. CQ polymerization also led to fusion of single bilayer vesicles at high lipid to protein ratio and to disassembly of bilayers at high C9 to lipid ratios. The data indicate that isolated C9 can be transformed from a water soluble monomeric protein to a membrane bound amphiphilic polymer. The membranolytic effect of polymerized C9 may be ascribed to the formation of a transmembrane protein channel in addition to physical effects on phospholipid bilayer structure, (Supported by the Swiss National Science Foundation, American Heart Association El No. 79-149 and USPHS Grants Ai 17354, CA 27489 and HL 16411.) COMPLEMENT ACTIVATION BY MYELIN: ACTIVATION OF CLASSICAL PATHWAY BY ISOLATED MYELIN MEMBRANES IN THE ABSENCE OF ANTIBODY. ~F~dmavafhy ~~ngu~~, Carol Koski and Moon Shin. Unversity of Maryland School of Medicine, Department of Pathology and Neurology, Baltimore, MD21201. Many pathological conditions of the central nervous system (CNS) involve damage to and removal of myelin membrane. Extensive phagocytosis of myelin by macrophages and astrocytes and release of myelin constituents into cerebrospinal fluid and into systemic circulation following CNS injury have been well documented. However, very little is known about the initiation of this membrane damage and mechanism of disposal of the damaged tissue. We are interested in the interaction between complement and myelin membranes and its’ possible role in the disposal of damaged myelin in vivo, because activation of complement generates both opsonin(s) and membrane attack complexes. In the present studies, we found that isolated rat or human CNS myelin consumes human complement in a dose dependent manner in the absence of specific antibodies, and it is about ten times more efficient than peripheral myelin. Activation of complement was demonstrated by showing C3 cleavage in the fresh serum incubated with myelin. CNS myelin incubated with C2 deficient (C2D) serum showed no complement consumption, and no factor B conversion was detected in the serum, thus excluding the alternative pathway activation. The involvement of the classical pathway was shown by the Cl transfer assay. Myelin incubated with C2D serum or with purified Cl, and washed, can transfer Cl activity to recipient cells, EAC4. A NEW CONCEPT OF INDUCING IMMUNE CYTOLYSIS: KILLING OF TUMOR CELLS VIA THE ALTERNATIVE PATHWAY INDUCED BY A COVALENT HYBRID OF MONOCLONAL ANTIBODY AND COBRA VENOM FACTOR. Car/-Wilhelm Vogel” and Hans J. MUller-Eberhard. Research Institute of Scripps Clinic, La Jolla, California. The unique specificity of monoclonal antibodies was combined with the cell killing function of the cytolytic alternative pathway of complement. The functionally CBb-like glycoprotein of cobra venom was linked to a murine monoclonal antibody directed to a human melanoma associated antigen by a disulfide bono using a heterobifunctional crosslinking reagent [N-succinimidyl-3-(2-pyridyldithio) propionate]. The covalent monoclonal antibody-cobra venom factor (CVF) complex exhibited specific cytolytic activity in the presence of normal or C4 deficient serum. It induced killing of melanoma cells but not of LG-2 lymphoblastoid cells or P815 mastocytoma cells. The cytolytic action of the monoclonal antibody-CVF complex was selective in that it was limited to the melanoma cells when these were in mixture with one of the other two cell lines. In absence of serum, the complex was noncytotoxic. The antibody or CVF alone had not cytolytic activity with or without serum. It is concluded that CVF covalently linked to melanoma cell bound antibody forms the stable C31C5 convertase with Factors B and D of the alternative pathway, which in turn causes formation of the membrane attack complex and thereby cell death. (Supported by USPHS Grants Al 17354, CA 27489, HL 16411 and DFG Fellowship No. 303/l.)