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Failure of complement regulation by the sickle erythrocyte results in increased membrane attack complex formation and reactive lysis
55
HETEROPOLYMER MEDIATED CLEARANCE OF TARGET ANTIGENS BOUND TO PRIMATE E R Y T H R O C Y T E S (E) V I A CR1. IN VIVO ANALYSES R.P. Taylora, C.J. Reist a, W.M. Schelda, D. Denny a, H-Y I_iangb, E. Martina auniversity of Virginia, Charlottesville, VA, USA; bChina-Japan Friendship Hospital, Peking, China We have investigated the feasibility of using cross-linked monoclonal antibodies (heteropolymers) for the clearance of target antigens from the circulation of primates. In the first phase of the work we used these heteropolymers (specific for primate E CR1 and the respective target antigen) to bind three different 1251.labelledantigens (human IgM, IgE and LDL) to 51Cr-labelled cynomolgus or rhesus monkey E. We then injected these doubly labelled E back into the animal's circulation. In all cases, there was rapid clearance (> 95% in less than 20 minutes) of the 1251-labelled proteins, but little, ff any clearance or lysis of the 51Cr-labelled E. In the second phase of the work we injected 12Sl-labelled human IgM into the circulation of a squirrel monkey. We established that over ca. 40 minutes the IgM circulated freely without clearance. We then infused an anti-lgM/antiCR1 heteropolymer (either unlabelled, or 1311-1abellecl) into the animal's circulation. In all experiments (5 different squirrel monkeys) we observed rapid binding of a substantial fraction of the IgM and heteropolymer to E followed by clearance of both labelled probes. Within 1 hour after heteropolymer injection, typically more than 70% of the IgM and heteropolymer were cleared. These protocols also led to a significant reduction in E CR1, followed by restoration of normal CR1 levels in ca. one week. This later finding is similar to that reported by Hebert's group (J. Immunol. 145: 4198, 1990) in experiments on immune complex clearance in primates. Although the details of the mechanism for heteropolymer-E mediated clearance of target antigens remain to be defined, it is likely that clearance follows a pathway similar to that operative in the E-mediated clearance of immune complexes. Our results provide additional evidence for the potential viability of using heteropolymers as a therapeutic.
E F F E C T OF A N T I S E N S E O L I G O N U C L E O T I D E S ON THE SECRETION OF C8 BY HepG2 CELLS. Francesco Tedesco#, Sabrina Perissutti#, Sabrina Mancardi* and Oscar Burrone*. #, Istituto di Patologia Generale, Universitd di Trieste, Trieste, Italy and *, International Centre for Genetic Engineering and Biotechnology, ICGEBUNIDO, Trieste, Italy C8 is synthesized as three separate chains, ct [3 and y, assembled into two subunits, ~-7 and 13 , which are secreted mostly as whole C8 molecule and only partially as free C80~-7. We have investigated the ability of the antisense oligonucleotides specific for each chain of C8 to inhibit the secretion of either the whole C8 or its subunits. The experiments were performed on HepG2 cells maintained in serum free medium containing 50 ~M of each oligonucleotide for 6-12 hr. The amount of C8 or C8 subunits secreted was measured by a sandwich ELISA using goat IgG to either ct-7 or as trapping antibodies, and biotin labeled goat IgG to whole C8 as revealing antibodies. The supernatants of the cell culture were also analyzed by SDS-PAGE under non-reducing conditions followed by Western blot to confirm the presence and relative amounts of the two subunits. The secretion of ct), was inhibited by 60-70% when the cells were treated with either C8ct or C8y antisense while it was not appreciably influenced by the C8~ antisense. On the other hand, the amount of C8~ released decreased by - 60% in the presence of the C8~l antisense and also by -40% when the C8a antisense was added to the cell cultures. These results suggest that the C8~ requires to be assembled into the complete C8 molecule in order to be secreted.
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Teixeira, J.E. I, Costa, R.S. 2, Lachmann, p.j.3, Wurzner, R. 3, Barbosa, J.E. I iDep. de Parasitologia, Microbiologia e Imunologia and Imunologia Cl{nica, Hospital das Cl{nicas, 2Dep.
de
Lab.
& de
Patologia,
Faculdade de Medicina de Ribeirao Preto, USP, Brazil, 3Molecular Immunopathology Unit, MRC Centre, Cambridge, England. The number of CRI in podocytes is reduced in nephropaties with severe glomerular damage, especially in the diffuse proliferative glomerulonephritis (DPGN) of Systemic Lupus Erythematosus (SLE). Individuals with SLE also present a decrease in CRI number in red blood cells associated with complement activation in vivo, with a simultaneous increase in C3dg on the surface of these cells. Considering that the reduction in podocyte C3b receptors may be due to a local proteolytic activity associated with activation of the complement system, we investigated the presence of TCC and CRl-stamp in histological sections of normal human kidneys and in biopsies from patients with SLE by the indirect immunoperoxidase technique. Intact CRI receptors were detected using TO5 monoclonal antibody as the first antibody. The CRl-stumps were detected using as first antibody a rabbit polyclonal antibody which recognizes a peptide corresponding to the CRI sequence between the proximal SCR and the transmembrane segment of the receptor. Immune TCC deposits were detected by the monoclonal WU 7-2 antibody which recognizes a neoepitope in C9. The results show different histological patterns in the glomerular TCC deposite which vary with the severity of glomerular damage. Less severe glomerular lesions present TCC deposits mainly in the mesangium (mesangial pattern). In lupus nephritis, in which the glomerular damage is more severe, TCC deposits were detected both in the mesangium and in capillary loops with podocyte involvement (mixed pattern). Patients with DPGN presented a marked reduction of intact podocyte CRI receptors in association with increased reactivity for the anti-Crl-stump antibody and with glomerular TCC deposits of mixed histological pattern. Two patients with DPGN with renal biopsies demonstrating strong reactivity for the anti-CRl-stump antibody presented intense PMN infiltrates in the glomeruli. These results suggest that the decrease in number of podocyte CRI receptors in severe glomerular lesions of SLE may be due to a local proteolytic activity associated with activation and deposition of the terminal components of complement.
FAILURE OF COMPLEMENT REGULATION BY THE SICKLE ERYTHROCYTE RESULTS IN INCREASED MEMBRANE ATTACK COMPLEX FORMATION AND REACTIVE LYSIS Samuel T. Test and Vicki S. Woolworth Children's Hospital Oakland Research Institute, Oakland, California USA Normal red cells induced to vesiculate by treatment with calcium and ionophore become sensitive to damage by activated complement (Test ST et al, Blood 78:3056, 1991). Because sickle cells release microvesicles as they circulate, we postulated that sickle cells might also be unusually sensitive to complement-dependent hemolysis. Complement activation is tightly regulated on the membrane of the normal erythrocyte, therefore defective complement regulation by the sickle cell would be necessary for complement-dependent hemolysis to occur. Sickle red blood cells (RBC) were fractionated according to density on discontinuous gradients of arabinogalactan and analyzed for sensitivity to complementmediated hemolysis. Increasing susceptbility to lysis by antibody and complement was apparent with increasing cell density, but there were minimal differences between AA and SS RBC. The densest fraction of sickle cells (and AA RBC) also exhibited decreases in immunoreactive CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) to 75% of the levels seen in the least dense fraction, levels ordinarily sufficient to sustain normal complement regulation. Nonetheless, a defect in the regulation of membrane attack complex formation in denser sickle erythrocytes was clearly apparent. The defect was characterized by increased binding of C5b-7 and of C9 to denser sickle cells and resulted in increased susceptibility of sickle cells to reactive lysis initiated by either C5b6 or activated cobra venom factor. Among the densest sickle cells, irreversibly sickled cells were especially sensitive to reactive lysis. Differences between SS and AA RBC disappeared when rabbit serum was substituted for human serum as a source of C8/C9 in C5b6-initiated reactive lysis, suggesting that CD59 function was subnormal. The similarity of this defect to that previously described in a patient with paroxysmal nocturnal hemoglobinuria suggests that complement-mediated hemolysis could play a role in the anemia of sickle cell disease.
HETEROPOLYMER MEDIATED CLEARANCE OF TARGET ANTIGENS BOUND TO PRIMATE E R Y T H R O C Y T E S (E) V I A CR1. IN VIVO ANALYSES R.P. Taylora, C.J. Reist a, W.M. Schelda, D. Denny a, H-Y I_iangb, E. Martina auniversity of Virginia, Charlottesville, VA, USA; bChina-Japan Friendship Hospital, Peking, China We have investigated the feasibility of using cross-linked monoclonal antibodies (heteropolymers) for the clearance of target antigens from the circulation of primates. In the first phase of the work we used these heteropolymers (specific for primate E CR1 and the respective target antigen) to bind three different 1251.labelledantigens (human IgM, IgE and LDL) to 51Cr-labelled cynomolgus or rhesus monkey E. We then injected these doubly labelled E back into the animal's circulation. In all cases, there was rapid clearance (> 95% in less than 20 minutes) of the 1251-labelled proteins, but little, ff any clearance or lysis of the 51Cr-labelled E. In the second phase of the work we injected 12Sl-labelled human IgM into the circulation of a squirrel monkey. We established that over ca. 40 minutes the IgM circulated freely without clearance. We then infused an anti-lgM/antiCR1 heteropolymer (either unlabelled, or 1311-1abellecl) into the animal's circulation. In all experiments (5 different squirrel monkeys) we observed rapid binding of a substantial fraction of the IgM and heteropolymer to E followed by clearance of both labelled probes. Within 1 hour after heteropolymer injection, typically more than 70% of the IgM and heteropolymer were cleared. These protocols also led to a significant reduction in E CR1, followed by restoration of normal CR1 levels in ca. one week. This later finding is similar to that reported by Hebert's group (J. Immunol. 145: 4198, 1990) in experiments on immune complex clearance in primates. Although the details of the mechanism for heteropolymer-E mediated clearance of target antigens remain to be defined, it is likely that clearance follows a pathway similar to that operative in the E-mediated clearance of immune complexes. Our results provide additional evidence for the potential viability of using heteropolymers as a therapeutic.
E F F E C T OF A N T I S E N S E O L I G O N U C L E O T I D E S ON THE SECRETION OF C8 BY HepG2 CELLS. Francesco Tedesco#, Sabrina Perissutti#, Sabrina Mancardi* and Oscar Burrone*. #, Istituto di Patologia Generale, Universitd di Trieste, Trieste, Italy and *, International Centre for Genetic Engineering and Biotechnology, ICGEBUNIDO, Trieste, Italy C8 is synthesized as three separate chains, ct [3 and y, assembled into two subunits, ~-7 and 13 , which are secreted mostly as whole C8 molecule and only partially as free C80~-7. We have investigated the ability of the antisense oligonucleotides specific for each chain of C8 to inhibit the secretion of either the whole C8 or its subunits. The experiments were performed on HepG2 cells maintained in serum free medium containing 50 ~M of each oligonucleotide for 6-12 hr. The amount of C8 or C8 subunits secreted was measured by a sandwich ELISA using goat IgG to either ct-7 or as trapping antibodies, and biotin labeled goat IgG to whole C8 as revealing antibodies. The supernatants of the cell culture were also analyzed by SDS-PAGE under non-reducing conditions followed by Western blot to confirm the presence and relative amounts of the two subunits. The secretion of ct), was inhibited by 60-70% when the cells were treated with either C8ct or C8y antisense while it was not appreciably influenced by the C8~ antisense. On the other hand, the amount of C8~ released decreased by - 60% in the presence of the C8~l antisense and also by -40% when the C8a antisense was added to the cell cultures. These results suggest that the C8~ requires to be assembled into the complete C8 molecule in order to be secreted.
DEPosn'IoN OF ~ wrm ~ P R ~ C~JJE[mtl~T OF t~tI'lqS °
OF A P
~
COtfl~$~ COMPLEX ( T e c ) I S A S S O C / A I ~ CR1 ~ t ~ ~ (CR1-STIR~) IN WITH
DIFFUSE
PROLIFERAXIVE
LUllS
Teixeira, J.E. I, Costa, R.S. 2, Lachmann, p.j.3, Wurzner, R. 3, Barbosa, J.E. I iDep. de Parasitologia, Microbiologia e Imunologia and Imunologia Cl{nica, Hospital das Cl{nicas, 2Dep.
de
Lab.
& de
Patologia,
Faculdade de Medicina de Ribeirao Preto, USP, Brazil, 3Molecular Immunopathology Unit, MRC Centre, Cambridge, England. The number of CRI in podocytes is reduced in nephropaties with severe glomerular damage, especially in the diffuse proliferative glomerulonephritis (DPGN) of Systemic Lupus Erythematosus (SLE). Individuals with SLE also present a decrease in CRI number in red blood cells associated with complement activation in vivo, with a simultaneous increase in C3dg on the surface of these cells. Considering that the reduction in podocyte C3b receptors may be due to a local proteolytic activity associated with activation of the complement system, we investigated the presence of TCC and CRl-stamp in histological sections of normal human kidneys and in biopsies from patients with SLE by the indirect immunoperoxidase technique. Intact CRI receptors were detected using TO5 monoclonal antibody as the first antibody. The CRl-stumps were detected using as first antibody a rabbit polyclonal antibody which recognizes a peptide corresponding to the CRI sequence between the proximal SCR and the transmembrane segment of the receptor. Immune TCC deposits were detected by the monoclonal WU 7-2 antibody which recognizes a neoepitope in C9. The results show different histological patterns in the glomerular TCC deposite which vary with the severity of glomerular damage. Less severe glomerular lesions present TCC deposits mainly in the mesangium (mesangial pattern). In lupus nephritis, in which the glomerular damage is more severe, TCC deposits were detected both in the mesangium and in capillary loops with podocyte involvement (mixed pattern). Patients with DPGN presented a marked reduction of intact podocyte CRI receptors in association with increased reactivity for the anti-Crl-stump antibody and with glomerular TCC deposits of mixed histological pattern. Two patients with DPGN with renal biopsies demonstrating strong reactivity for the anti-CRl-stump antibody presented intense PMN infiltrates in the glomeruli. These results suggest that the decrease in number of podocyte CRI receptors in severe glomerular lesions of SLE may be due to a local proteolytic activity associated with activation and deposition of the terminal components of complement.
FAILURE OF COMPLEMENT REGULATION BY THE SICKLE ERYTHROCYTE RESULTS IN INCREASED MEMBRANE ATTACK COMPLEX FORMATION AND REACTIVE LYSIS Samuel T. Test and Vicki S. Woolworth Children's Hospital Oakland Research Institute, Oakland, California USA Normal red cells induced to vesiculate by treatment with calcium and ionophore become sensitive to damage by activated complement (Test ST et al, Blood 78:3056, 1991). Because sickle cells release microvesicles as they circulate, we postulated that sickle cells might also be unusually sensitive to complement-dependent hemolysis. Complement activation is tightly regulated on the membrane of the normal erythrocyte, therefore defective complement regulation by the sickle cell would be necessary for complement-dependent hemolysis to occur. Sickle red blood cells (RBC) were fractionated according to density on discontinuous gradients of arabinogalactan and analyzed for sensitivity to complementmediated hemolysis. Increasing susceptbility to lysis by antibody and complement was apparent with increasing cell density, but there were minimal differences between AA and SS RBC. The densest fraction of sickle cells (and AA RBC) also exhibited decreases in immunoreactive CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) to 75% of the levels seen in the least dense fraction, levels ordinarily sufficient to sustain normal complement regulation. Nonetheless, a defect in the regulation of membrane attack complex formation in denser sickle erythrocytes was clearly apparent. The defect was characterized by increased binding of C5b-7 and of C9 to denser sickle cells and resulted in increased susceptibility of sickle cells to reactive lysis initiated by either C5b6 or activated cobra venom factor. Among the densest sickle cells, irreversibly sickled cells were especially sensitive to reactive lysis. Differences between SS and AA RBC disappeared when rabbit serum was substituted for human serum as a source of C8/C9 in C5b6-initiated reactive lysis, suggesting that CD59 function was subnormal. The similarity of this defect to that previously described in a patient with paroxysmal nocturnal hemoglobinuria suggests that complement-mediated hemolysis could play a role in the anemia of sickle cell disease.