- Email: [email protected]
How are immune complexes bound to the primate erythrocyte complement receptor transferred to acceptor phagocytic cells?
Molecular Immunology 36 (1999) 827±835
www.elsevier.com/locate/molimm
How are immune complexes bound to the primate erythrocyte complement receptor transferred to acceptor phagocytic cells? Alessandra Nardin, Margaret A. Lindorfer, Ronald P. Taylor* Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
Abstract Immune complexes (IC) bound to the primate erythrocyte (E) complement receptor (CR1) are cleared from the circulation of primates and localized to phagocytic cells in the liver and spleen without E destruction. IC can be bound to E CR1 either via C3b opsonization or with cross-linked mAb complexes (heteropolymers, HP) which contain a mAb speci®c for CR1 and a mAb speci®c for an antigen. The long-term goal of our work is to apply the HP system to the treatment of human diseases associated with blood-borne pathogens. This review discusses the mechanism by which the E-bound IC are transferred to acceptor cells. Our studies in animal models as well as our in vitro investigations indicate that IC transfer is rapid (usually >90% in 10 min) and does not lead to lysis or phagocytosis of the E. Experiments with speci®c inhibitors and the use of IC prepared with Fab ' fragments suggest that transfer depends mainly upon recognition by Fc receptors on the acceptor cell. Moreover, we ®nd that IC release from the E is associated with a concerted loss of CR1, and is followed by uptake and internalization of the IC by the acceptor cell. We suggest that recognition and binding of the E-bound IC substrates by Fc receptors allows close contact between the E and acceptor cells, which in turn facilitates proteolysis of E CR1, presumably by a macrophage-associated protease. After proteolysis, the released IC are internalized by the macrophages. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Bispeci®c antibodies; Erythrocytes; Immune complexes; Complement receptor 1; Complement
1. Introduction Since the original description of the preparation of monoclonal antibodies (mAbs), much eort has focused on development of mAb-based approaches to treating human diseases (Burnie and Matthews, 1998; Kohler and Milstein, 1975; Multani and Grossbard, 1999; Waldmann and O'Shea, 1999). The long term goal of our work is to develop a general platform immunotherapy for treatment of human diseases associated with blood-borne pathogens (Taylor et al., 1991, 1997a). Fundamental to our approach is the hypothesis that it should be possible to facilitate the safe and rapid clearance of pathogens from the circulation * Corresponding author. Tel.: +1-804-924-2664; fax: +1-804-9245069. E-mail address: [email protected] (R.P. Taylor).
by utilizing and enhancing the paradigm of a natural process, the primate erythrocyte (E) immune complex (IC) clearance reaction (Kuhn et al., 1998; Cornaco et al., 1983; Cosio et al., 1990; Taylor et al., 1991). This reaction is mediated by the E complement receptor (CR1), an intrinsic membrane glycoprotein with speci®city for C3b, a protein fragment generated during complement activation (Fearon, 1980). Complement-opsonized IC containing covalently incorporated C3b form in the circulation and bind to primate E via CR1 in the immune adherence (IA) reaction (Nelson, 1953, 1955). The IC are then safely cleared from the circulation and neutralized in the liver without E destruction (Cornaco et al., 1983; Cosio et al., 1990; Davies, 1996; Davies et al., 1990; Schierli and Taylor, 1989). In numerous in vitro and in vivo models, we have demonstrated that, by making use of bispeci®c mAb
0161-5890/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 9 9 ) 0 0 1 0 3 - 0
828
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
complexes (heteropolymers, HP) which contain a mAb speci®c for CR1 cross-linked with a mAb speci®c for an antigenic site on a target pathogen, we can ensure high anity binding of any target pathogen to E (Nardin et al., 1998; Reist et al., 1993; Taylor et al., 1991, 1997a). The anti-CR1 mAb acts as a surrogate for C3b, and binding is achieved independent of complement opsonization. Moreover, our in vivo experiments in monkeys and in mice have demonstrated that prototype pathogens bound to E CR1 via the HP system are rapidly cleared from the circulation and localized to the liver, thus con®rming our original hypothesis (Nardin et al., 1999; Reist et al., 1993, 1994; Taylor et al., 1997b). Another goal of this work is to obtain a detailed understanding of the basic process by which natural C3b-opsonized and E-bound IC are transferred from E to acceptor phagocytic cells. We believe that the mechanism of this transfer reaction is general and virtually identical for substrates bound to E CR1 via either C3b or HP. The transfer reaction requires the presence of the Fc portions of the antibodies in the IC and results in the concerted loss from E of the CR1 engaged in IC binding. We suggest that close contact between E and acceptor cells, facilitated by Fc receptors, allows one or more membrane associated proteases on the phagocyte to proteolytically cut E CR1 and release it, together with the C3b- or HP-opsonized complex, for subsequent internalization. It is possible that other receptors (e.g., CR3 or CD14 (Wright, 1995; Wright and Jong, 1986)) on the acceptor cell may engage the E-bound IC to allow for the proteolysis and internalization step. However, any release of IC from E in the circulation mediated by Factor I, presumably cleaving C3b to C3bi and then C3dg (Medof et al., 1982), is unlikely to play a signi®cant or direct role in the transfer reaction. We will present evidence to support our hypothesis for the proteolytic mechanism.
Fig. 1. Absence of sequestration of IC-opsonized erythrocytes. 51Crlabeled autologous erythrocytes, with in vitro prebound antibody/125I-dsDNA IC, were infused iv in a chimpanzee and counts were monitored in the circulation. Data are presented as the percentage of counts remaining relative to 1 min values (mean2SD; n = 4). By 10 min, >75% of initially E-bound IC had been ``stripped'' from E, but < 2% of these counts were detectable in the total plasma fraction. By 30 min, 95% of IC had been stripped from E with 06% of counts in the total plasma fraction (1.4% trichloroacetic acid precipitable and 4.5% nonprecipitable). No free 51Cr (<0.2%) was detectable in the plasma samples.
cleared from the circulation without loss of E; >75% of the IC were removed from the E by 10 min and 95% by 30 min (Fig. 1). Our in vitro investigations demonstrated that Factor I mediated-release from E of these complement-opsonized IC is quite slow (Fig. 2)
2. Immune complexes bound to CR1 via C3b or HP can be rapidly and speci®cally cleared from the circulation We have observed that, during in vivo clearance of IC bound to E CR1, the IC themselves, but not the E, are removed from the circulation and localized to the liver. This has held true for IC bound to CR1 either via C3b opsonization or via HP. We performed a series of ex-vivo experiments in which 125I-labeled IC were bound in vitro to 51Cr labeled primate E, reinfused into non-human primates or mice, and then followed in the bloodstream. In experiments in chimpanzees, complement (C3b)-opsonized IgG antidsDNA/125I labeled-dsDNA IC were bound in vitro to autologous 51Cr labeled E and re-infused into the animals (Kimberly et al., 1989). The IC were rapidly
Fig. 2. Release of erythrocyte-bound IC in serum-EDTA. Ab/3Hlabeled dsDNA IC were bound to erythrocytes obtained from several primates (chimpanzee (CH), baboon (BA), rhesus (RH) and cynomolgus (CY) monkeys, human (HU)), and then autologous serum +0.01 M EDTA was added and the percentage of IC released after 1 h was determined. The mean2SD for three independent determinations (with cells and serum from dierent animals) is shown.
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
Fig. 3. Clearance in a rhesus monkey of 125I-labeled IgG antidsDNA antibodies prebound ex vivo via an antigen-based heteropolymer (AHP) to autologous 51Cr-labeled erythrocytes. 51Cr counts in the plasma averaged <2% of erythrocyte-associated counts. The 51 Cr counts after 80 min are approximately twice those for the ®rst 80 min, re¯ecting the cumulative eect of the 2 infusions. Little, if any, 51Cr was cleared. The ®rst line for 125I-IgG (IgG anti-dsDNA from SLE patient Ma) is the theoretical ®t to the data based on double-exponential decay, the second (IgG anti-dsDNA from SLE patient Ha) represent the theoretical ®t to a single exponential decay.
(Edberg et al., 1992). This dierence in release kinetics for the in vivo (fast) vs the in vitro (slow) system indicated that Factor I-mediated degradation of C3b to C3bi and then to C3dg (which does not bind to CR1 (Medof et al., 1982)) is too slow to account for the rapid transfer reaction observed in vivo. Ex-vivo experiments using both HP and antigenbased heteropolymers (AHP) showed a similar pattern of clearance of E-bound IC. In rhesus monkeys, both 125 I labeled proteins prebound to 51Cr labeled E via HP (Reist et al., 1993) and 125I labeled IgG antidsDNA prebound to 51Cr labeled E via an antigenbased HP (anti-CR1 mAb cross linked with dsDNA) (Fig. 3, Ferguson et al., 1995) were cleared from the circulation with similar kinetics (090% in 10 min), and without loss of E. Experiments in A/J mice provided evidence for the generality of the mechanism of the transfer reaction. 125I-labeled bacteriophage FX174 was bound ex-vivo via HP to 51Cr labeled primate E, and, upon infusion into the mouse, >90% of FX174 cleared to the liver in 10 min (Nardin et al., 1999). In both the HP and AHP systems, C3b is not utilized to facilitate E binding. Thus, it is unlikely that factor I (which cleaves C3b but not IgG (Medof et al., 1982)) can play a role in the release of substrates bound to E CR1 via anti-CR1 mAbs in the HP and AHP systems.
829
Fig. 4. IC formed in situ on primate E CR1 are cleared from the circulation coincident with loss of CR1. 125I-labeled anti-CR1 mAb 7G9 (0.8 mg) was infused iv into the circulation of a monkey (5.3 kg), and more than 75% of the infused reagent bound to E. At 61.5 min (see arrow) a bolus of monkey antibodies to mouse IgG was infused. The E pellets were counted to measure bound 125Ilabeled 7G9, and probed with additional 125I-labeled anti-CR1 mAbs 7G9 and HB8592 (initially 2900 and 1140 epitopes per E, respectively) to determine total E CR1. The increase in apparent CR1 epitopes after the infusion at 61.5 min is due to ``capture'' of the mouse mAb probes by the monkey anti-mouse IgG bound to the E. More than 90% of the plasma counts associated with the infused 125Ilabeled mAb 7G9 were also cleared from the circulation by the end of the experiment.
3. In vivo transfer of IC from primate E (or, in nonprimates, platelets) to phagocytic cells involves removal of cell-associated CR1. Historical precedence and recent studies In 1975, Miller et al. reported that platelets of old NZB/NZW mice with chronic IC disease due to a lupus-like syndrome had substantially lost their ability to bind complement-opsonized IC infused into the circulation. In mice and other non-primates IA activity is platelet associated, and it was suggested that ``receptors . . . on the surface membrane of cells from old mice might not be available for binding'' (Miller et al., 1975). This prescient observation is supported by more recent experiments in monkey models and in humans indicating that processing of complement-®xing IC is correlated with a decrease in levels of E CR1, the primate IA receptor (Cosio et al., 1990; Davies, 1996; Davies et al., 1990, 1992). Moreover, E CR1 levels are reduced in individuals with diseases characterized by chronic IC processing such as the auto-immune disease systemic lupus erythematosus (SLE) and AIDS (Fearon, 1985; Iida et al., 1982; Lach-Tri®lie et al., 1999; Ross et al., 1985a; Schierli and Taylor, 1989; Tausk et al., 1986). The parallel between the primate and non-primate systems is therefore clear.
830
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
Fig. 5. Loss of bacteriophage FX174 (black triangles), HP (anti-CR1 mAb 1B4 X anti-FX174 mAb 7B7; black squares) and CR1 (black circles) from biotinylated human E infused iv in the circulation of an A/J mouse as determined by ¯ow cytometry. Approximately 10% of the blood cells were positive for the phycoerythrin-labeled streptavidin used to identify the human E, with virtually no loss of these E from the circulation of the mouse during the experiment. Fluorescein isothiocyanate (FITC)-labeled mAb 7G9, not competing with mAb 1B4 present in the HP, was used to probe for CR1. In a parallel control, the same biotinylated human E (not opsonized with HP and FX174) were infused in another A/J mouse and showed virtually no loss of CR1 (white circles).
We demonstrated loss of E CR1 in a squirrel monkey in a study of HP-mediated clearance of human IgM, which was selected as a prototype pathogen (Reist et al., 1994). First human IgM, then HP (anti-CR1 mAb cross-linked with anti-human IgM mAb) were infused into the circulation of the monkey. Separate radiolabels on the HP and IgM allowed us to demonstrate that the HP facilitated binding of human IgM to the monkey E. Soon after HP infusion and binding of the substrate to E, both HP and IgM were cleared from the circulation and localized to the liver. Moreover, coincident with clearance of these E-bound substrates, CR1 levels on the monkey E also decreased considerably (Reist et al., 1994). We sought to generalize the clearance reaction by selecting a simpler model: IC were formed directly on E CR1 by infusing ®rst large amounts of 125I-labeled anti-CR1 mouse mAb 7G9, then monkey anti-mouse IgG (Fig. 4) (Taylor et al., 1997c). The IC does indeed form on the E, and it is cleared together with CR1 in a concerted reaction. As mentioned above, in a mouse model we found that the bacteriophage FX174 is cleared to the liver if it is infused bound to primate E via HP. Flow cytometric analyses of this reaction demonstrate simultaneous loss of FX174, HP, and CR1 from the population of
human E infused into the mouse (Fig. 5) (Nardin et al., 1999). Independent studies by Ulevitch and Frank demonstrated that infusion of LPS into the bloodstream of rabbits or guinea pigs leads to a rapid but transient loss of platelets from the bloodstream, in a reaction which requires complement activation (Kane et al., 1973; Mathison and Ulevitch, 1979, 1981). They reported that, immediately after infusion, a large fraction of the LPS is bound to platelets. Furthermore, the majority of the LPS is rapidly cleared from the circulation and localized to the liver. It is likely that IA of C3b-opsonized LPS/IgG IC to platelets (mediated by endogenous antibodies and complement) causes the platelets to bind transiently to Fc receptors on ®xed tissue macrophages in the liver. Proteolysis and removal of the platelets' IA receptors containing bound IC would allow these cells to be released back into the bloodstream. It is important to note that the platelets which reappear in the circulation no longer bind LPS, although they do bind LPS after it is ®rst infused (Kane et al., 1973; Mathison and Ulevitch, 1979, 1981). These platelets should have severely reduced IA activity if tested with complement-opsonized substrates, compared to platelets isolated from the animals before the experiment. Removal of the IA receptor from non-primates' platelets or of CR1 from primate E appears to be a necessary condition for transfer of cell-bound IC to phagocytic cells in the liver. We suggest that close association of the E-bound IC with ®xed tissue macrophages (mediated by Fc and possibly other receptors) is followed by a proteolytic cut of CR1. Phagocytic cells express numerous membrane associated proteases (Bauvois et al., 1992), and E CR1 is quite sensitive to proteolysis and can easily be removed from E by a variety of proteases (Pascual et al., 1994). Several membrane-associated proteins, including CR2, L-selectin, IL-6R, TNF-aR, and CD16, can be shed from cells by protease activity. In some cases, a Zn-dependent metalloprotease, not susceptible to common protease inhibitors, has been found responsible for the proteolytic cleavage (Chen et al., 1995; FremeauxBacchi et al., 1996; Huizinga et al., 1990; Mohler et al., 1994; Mullberg et al., 1995). In fact, L-selectin and CR2 each contain multiple short consensus repeat motifs (Ahearn and Fearon, 1989) similar to those expressed on CR1, and are released from leukocytes and lymphocytes, respectively, by cleavage in a domain proximal to the transmembrane portion. Recently Hamer et al. (1998) have studied COS cells transfected with membrane-bound CR1. Their work suggests that CR1 can be released after a proteolytic cut within the transmembrane region. In these reactions a protein is ``shed'' from the cell due to a protease present on the cell itself. However, in the case of E CR1, it is likely
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
Fig. 6. Infused Fab' X Fab' HP does not mediate clearance unless followed by intact IgG anti-FX174 mAb. At time zero, 100 mg of 131 I-labeled bacteriophage FX174 was infused into the circulation of a 8.7 kg monkey, and the Fab' X Fab' HP was infused 48 min later. Although a very high level of E binding accompanied infusion of the HP, E-bound counts did not clear from the circulation, and liver counts remained ¯at. Forty min later (see arrow on the x axis), whole anti-FX174 mAb 7B7 was infused, and, after a 5-min delay, clearance commenced and proceeded rapidly. Anger camera was used for imaging: background (before infusion) was 2000 counts, liver background was 130 counts, and a total of 31,000 counts were infused.
that the protease(s) which causes release of E CR1 is provided by the phagocytic cell, and not by the E. We propose that a Zn-dependent metalloprotease provided by the acceptor cell can cause release of E CR1, and our preliminary studies (Craig and Taylor, manuscript in preparation) support this mechanism. An extensive literature documents the importance of anti-E antibodies in autoimmune hemolytic anemias (Schreiber and Frank, 1972; Victoria et al., 1990). Opsonization of E with antibodies against surface epitopes has traditionally been used to generate model substrates for in vitro study of phagocytosis and for in vivo investigation of the mechanisms by which antibody-opsonized particles are removed from the bloodstream (Edberg and Kimberly, 1992; Frank, 1989; Schreiber and Frank, 1972). In view of these facts, it is surprising that the primate E can serve as an IC carrier without itself being destroyed. We believe that E CR1 presents a possibly unique and privileged site on the E surface. Complement-opsonized IC containing IgG can indeed bind naturally to E via CR1 without causing E destruction. We have demonstrated that binding of large amounts of IgG to CR1 on primate E does not lead to phagocytosis of the E by monocytes (Reinagel et al., 1997). Instead, when IgG is bound to other sites on E, the cells are indeed phagocytosed. These disparate observations are reconciled by allowing for pro-
831
Fig. 7. Clearance in A/J mice of 125I-labeled bacteriophage FX174 bound ex vivo to 51Cr-labeled human E via Fab' HP and then infused iv into the circulation of the mouse (black squares). Data are presented as the percentage of 125I counts remaining bound to the 51 Cr-labeled E. In one case FX174 was bound to the E and then further opsonized with anti-FX174 mAb 7B7 before injection into the mouse (black circles).
teolysis of CR1. If all E-associated IgG is bound to CR1, which is then released by proteolytic cleavage from the E, naturally the E will be spared, as the opsonizing reagent (IgG) will be removed. This mechanism should work equally well if IC are bound to CR1 via C3b, the natural ligand, or via the anti-CR1 mAb in the HP.
4. Intact mAbs are required for E CR1 bound IC recognition and transfer to phagocytic cells. The role of Fc and other receptors Both Fc receptors and complement are likely to participate in the transfer reaction. In applying the HP system in both monkey and mouse models, we found that the presence of intact IgG on the E-bound IC was an absolute requirement for ecient and rapid transfer. In several experiments we observed rapid E binding and clearance of bacteriophage FX174 through an anti-FX174 X anti-CR1 HP. However, Fig. 6 shows that an Fab ' X Fab ' HP administered to a monkey after infusion of 131I labeled FX174 was sucient to ensure quantitative binding of bacteriophage to E but unable to facilitate clearance (Taylor et al., 1997b). Only after an intact anti-FX174 mAb was injected into the circulation (and bound to other sites on the Ebound substrate) were counts cleared from the E and localized to the liver. Similarly, in mice, clearance of
832
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
125
I labeled FX174 bound to E (human or monkey) via an Fab ' based HP was slow. Additional binding of an intact anti-FX174 mAb to the E-Fab ' HP-FX174 complex restored the normal clearance pattern (Fig. 7) (Nardin et al., 1999). The need for the Fc portion of the mAbs could re¯ect engagement of Fc receptors as well as a requirement for complement activation and opsonization of the IC, with subsequent involvement of complement receptors such as CR1 or CR3 on the phagocytes (Ahearn and Fearon, 1989; Hermanowski-Vosatka et al., 1988; Ross et al., 1985b). In the mouse model, interference with complement activation at the C3b opsonization step, through use of either cobra venom factor or mouse complement regulatory protein Crry, resulted in impaired clearance of E-bound FX174 (Nardin et al., 1999). However, in vitro studies of transfer of HP-bound FX174 from E to the murine macrophage cell line P388D1 showed that ecient transfer occurred in the absence of complement (Reinagel and Taylor, submitted for publication). An in vitro system which focused on transfer of E. coli, bound to E via HP, to acceptor monocytes provides further insight (Kuhn et al., 1998). Blockade of actin polymerization with cytochalasin D inhibited internalization, but use of colchicine (which blocks microtubule formation) did not inhibit transfer. Since both FcR and CR1 require intact actin ®laments for phagocytosis (Greenberg and Silverstein, 1993; Newman et al., 1991) but CR1 also requires microtubule assembly, internalization in our in vitro model is apparently FcR mediated. Other receptors may play a role in transfer, depending upon the nature of the substrate bound to E CR1. For example, CD14, CR3, and the mannose receptor can bind LPS and other bacterial structures (Stahl and Ezekowitz, 1998; Wright, 1995; Wright and Jong, 1986): interaction of these receptors with bacteria bound to E via HP could allow for close juxtaposition of the respective cells, followed by proteolysis of CR1 and release of bacteria. In fact, we found that blockade of Fc receptors alone only weakly blocked monocyte mediated release from E of HP-bound E. coli (Kuhn et al., 1998). However, uptake of the E. coli by monocytes was inhibited more eectively. Thus, close and sustained contact between E and phagocyte, even if mediated by receptors unable to support internalization, could be sucient for cleavage of CR1 and removal of the CR1-HP-Ag complex from E. CR3 binds LPS in an EDTA-inhibitable fashion and independent from C3bi opsonization (Wright, 1995; Wright and Jong, 1986). Although EDTA signi®cantly blocked uptake of E. coli by the monocytes, it seems unlikely that CR3 alone was responsible for the complete transfer reaction, since in the absence of HP the bacteria were weakly taken up by the monocytes.
Fig. 8. Clearance from the circulation of monkeys of 125I-labeled E. coli prebound ex vivo to autologous 51Cr-labeled E via either C3b opsonization (autologous serum was used) or an HP made with antiCR1 mAb 7G9 cross linked with anti-E. coli mAb 44. Monkey A (rhesus) was infused ®rst with E-HP-E. coli, then, at 51 min, with EC3b-E. coli. Monkey B (stump-tailed macaque) was infused ®rst with E-C3b-E. coli, then, at 51 min, with E-HP-E. coli; for the experiment in monkey B, 2 times more 125I-labeled E. coli were bound to 5 times less 51Cr-labeled E.
5. Direct comparisons of the transfer reaction for IC bound to E via C3b or via the HP (or AHP) systems The key presumption in our approach is that, with respect to the transfer reaction, IC bound to E via anti-CR1 mAbs (HP and AHP) will be processed in the same fashion as natural C3b-opsonized IC bound to E. Our recent work in an in vitro model, on the prototypical IC dsDNA/IgG anti-dsDNA, con®rms this hypothesis. Since our studies on this IC system 10 years ago (Kimberly et al., 1989) ®rst lead to our working hypothesis on the mechanism of the transfer reaction and to the development of HP and AHP, it is reassuring that the present studies continue to be consistent with the original postulate. We used the same SLE IgG anti-dsDNA antibodies to prepare both types of E-associated IC, and then studied transfer to U937 cells. Both C3b-opsonized dsDNA/anti-dsDNA IC and the AHP-anti-dsDNA IC, after ®rst binding to E, are transferred to U937 cells with similar and rapid kinetics. Moreover, monomeric mouse IgG2a, which
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
binds well to the high avidity Fc receptor, inhibits transfer of both types of IC, and transfer of the AHP complex is accompanied by loss of E CR1 (Craig and Taylor, abstract submitted). We performed an ex-vivo study to examine clearance of E. coli in monkeys after it is bound to E. In this experiment 125I-labeled E. coli was bound in vitro to 51 Cr-labeled monkey E, either via complement opsonization with autologous monkey serum or with a speci®c HP (Kuhn et al., 1998). The doubly-labeled substrates were then consecutively infused into the circulation of two monkeys, and the rate of clearance of infused materials was measured. Fig. 8 indicates that the clearance kinetics for the two substrates are quite similar. E-bound 125I-labeled E. coli is cleared quickly in both cases, with >95% clearance in less than 10 min. There is no loss of the E that had bound C3bopsonized bacteria, although there is a small but transient loss from the circulation of the E containing HP and E. coli. This experiment, with a dierent substrate from the antibody/dsDNA IC, provides further evidence that IC bound to E are handled in a similar fashion, whether they are bound via C3b opsonization or via a HP construct in which the mAb to CR1 serves as a surrogate for C3b. This precludes a role for factor I in the transfer reaction and is in agreement with earlier in vitro investigations in other model systems (Emlen et al., 1989, 1992). 6. Future considerations Several infectious agents are known to express ligands speci®c for receptors present on cells belonging to the reticuloendothelial system (RES), so that entrance results in productive infection instead of destruction of the pathogen (e.g., HIV and Marburg virus (Becker et al., 1995; Fauci, 1996; Monte®ori, 1997)). However, there are precedents for the use of bispeci®c antibody complexes in redirecting the pathogen through Fc receptors into a cell compartment where it can be eciently killed (Connor et al., 1991). One of the major challenges for our future work will be to determine if use of the HP-E system will allow for presentation of bona®de pathogens to the RES under conditions which do indeed lead to neutralization and destruction of the pathogens. Acknowledgements This work was supported by the Defense Advanced Research Projects Agency, Order MDA972-96-K-001 under contract MDA972-96-K-003, and by NIH grant AR43307. The authors would like to thank Dr P. Klebba for kindly providing the E. coli and speci®c
833
mAb, and Drs W. Emlen and J. Edberg for stimulating discussions. Figs. 1±7 were all reproduced with permission from the respective journals. Fig. 1: The Journal of Clinical Investigation (Kimberly et al., 1989). Figs. 2, 5 and 7: The European Journal of Immunology (Edberg et al., 1992; Nardin et al., 1999). Fig. 3: Arthritis and Rheumatism (Ferguson et al., 1995). Fig. 4: Clinical Immunology and Immunopathology (Taylor et al., 1997c). Fig. 6: The Journal of Immunology (Taylor et al., 1997b. Copyright 1997. The American Association of Immunologists).
References Ahearn, J.M., Fearon, D., 1989. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46, 183±219. Bauvois, B., Sanceau, J., Wietzerbin, J., 1992. Human U937 cell surface peptidase activities: characterization and degradative eect on tumor necrosis factor. Eur. J. Immunol. 22, 923±930. Becker, S., Spiess, M., Klenk, H., 1995. The asialoglycoprotein receptor is a potential liver-speci®c receptor for Marburg virus. J. Gen. Virol. 76, 393±399. Burnie, J.P., Matthews, R.C., 1998. The renaissance of antibody therapy. J. Antimicrob. Chemother. 41, 319±322. Chen, A., Engel, P., Tedder, T.F., 1995. Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. J. Exp. Med. 183, 519±530. Connor, R.I., Dinces, N.B., Howell, A.L., Romet-Lemonne, J.L., Pasquali, J.L., Fanger, M.W., 1991. Receptors for IgG on human monocytes and macrophages are not infectivity receptors for human immunode®ciency virus type 1: studies using bispeci®c antibodies to target HIV-1 to various myeloid cell surface molecules including the FcR. Proc. Natl. Acad. Sci. USA 88, 9593± 9597. Cornaco, J.B., Hebert, L.A., Smead, W.L., Van Aman, M.E., Birmingham, D.J., Waxman, F.J., 1983. Primate erythrocyteimmune complex-clearing mechanism. J. Clin. Invest. 71, 236± 247. Cosio, F.G., Xiao-Ping, S., Birmingham, D.J., Van Aman, M., Hebert, L.A., 1990. Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates. J. Immunol. 145, 4198±4206. Davies, K.A., 1996. Complement, immune complexes and systemic lupus erythematosus. Brit. J. Rheum. 35, 5±23. Davies, K.A., Hird, V., Stewart, S., Sivolapenko, G.B., Jose, P., Epenetos, A.A., et al., 1990. A study of in vivo immune complex formation and clearance in man. J. Immunol. 144, 4613±4620. Davies, K.A., Peters, M., Beynon, H.L., Walport, M.J., 1992. Immune complex processing in patients with systemic lupus erythematosus. J. Clin. Invest. 90, 2075±2083. Edberg, J.C., Kimberly, R.P., Taylor, R.P., 1992. Functional characterization of non-human primate erythrocyte immune adherence receptors: implications for the uptake of immune complexes by the cells of the mononuclear phagocytic system. Eur. J. Immunol. 22, 1333±1339. Edberg, J.C., Kimberly, R.P., 1992. Receptor speci®c probes for the study of Fc receptor speci®c function. J. Immunol. Meth. 148, 179±187. Emlen, W., Burdick, G., Carl, V., Lachmann, P.J., 1989. Binding of
834
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
model immune complexes to erythrocyte CR1 facilitates immune complex uptake by U937 cells. J. Immunol. 142, 4366±4371. Emlen, W., Carl, V., Burdick, G., 1992. Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes. Clin. Exp. Immunol. 89, 8±17. Fauci, A.S., 1996. Host factors and the pathogenesis of HIV-induced disease. Nature 384, 529±534. Fearon, D.T., 1980. Identi®cation of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152, 20±30. Fearon, D.T., 1985. Human complement receptors for C3b (CR1) and C3 d (CR2). J. Invest. Dermatol. 85, 53±57. Ferguson, P.J., Reist, C.J., Martin, E.N., Johnson, C., Greene, K.L., Kuhn, S., et al., 1995. Antigen-based heteropolymers: a potential therapy for binding and clearing autoantibodies via erythrocyte CR1. Arth. Rheumat. 38, 190±200. Frank, M.M., 1989. The role of macrophages in bloodstream clearance. In: Human Monocytes. Academic Press, New York, pp. 337±344. Fremeaux-Bacchi, V., Bernard, I., Maillet, F., Mani, J., Fontaine, M., Bonnefoy, J., et al., 1996. Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-barr virus receptor, CR2) that binds iC3b and CD23. Eur. J. Immunol. 26, 1497±1503. Greenberg, S.C.P., Silverstein, S.C., 1993. Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 177, 529±534. Hamer, I., Paccaud, J.-P., Belin, D., Maeder, C., Carpentier, J.-L., 1998. Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain. Biochem. J. 329, 183±190. Hermanowski-Vosatka, A., Detemers, P.A., Gotze, O., Silverstein, S.C., Wright, S.D., 1988. Clustering of ligand on the surface of a particle enhances adhesion to receptor-bearing cells. J. Biol. Chem. 263, 17,822±17,827. Huizinga, T.W.J., de Haas, M., Kleijer, M., Nuijens, J.H., Roos, D., von dem Borne, A.E.G.Kr, 1990. Soluble Fc receptor III in human plasma originates from release by neutrophils. J. Clin. Invest. 86, 416±423. Iida, K., Mornaghi, R., Nussenzweig, V., 1982. Complement receptor (CR1) de®ciency in erythrocytes from patients with systemic lupus erythematosus. J. Exp. Med. 155, 1427±1438. Kane, M.A., May, J.E., Frank, M.M., 1973. Interactions of the classical and alternative complement pathway with endotoxin lipopolysaccharide. J. Clin. Invest. 52, 370±376. Kimberly, R.P., Edberg, J.C., Merriam, L.T., Clarkson, S.B., Unkeless, J.C., Taylor, R.P., 1989. In vivo handling of soluble complement ®xing Ab/dsDNA immune complexes in chimpanzees. J. Clin. Invest. 84, 962±970. Kohler, G., Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of prede®ned speci®city. Nature 256, 495±497. Kuhn, S.E., Nardin, A., Klebba, P.E., Taylor, R.P., 1998. Escherichia coli bound to the primate erythrocyte complement receptor via bispeci®c monoclonal antibodies are transferred to and phagocytosed by human monocytes in an in vitro model. J. Immunol. 160, 5088±5097. Lach-Tri®lie, E., Marfurt, J., Schwarz, S., Sadallah, S., Schierli, J.A., 1999. Complement receptor 1 (CD35) on human reticulocytes: normal expression in systemic lupus erythematosus and HIV-infected patients. J. Immunol. 162, 7549±7554. Mathison, J.C., Ulevitch, R.J., 1979. The clearance, tissue distribution, and cellular localization of intravenously injected lipopolysaccharide in rabbits. J. Immunol. 123, 2133±2143. Mathison, J.C., Ulevitch, R.J., 1981. In vivo interaction of bacterial lipopolysaccharide (LPS) with rabbit platelets: modulation by C3 and high density lipoproteins. J. Immunol. 126, 1575±1580. Medof, M.E., Prince, G.M., Oger, J.J.F., 1982. Kinetics of inter-
action of immune complexes with complement receptors on human red blood cells: modi®cation of complexes during interaction with red cells. Clin. Exp. Immunol. 48, 715±725. Miller, G.W., Steinberg, A.D., Green, I., Nussenzweig, V., 1975. Complement-dependent alterations in the handling of immune complexes by NZB/W mice. J. Immunol. 114, 1166±1170. Mohler, K.M., Sleath, P.R., Fitzner, J.N., Cerretti, D.P., Alderson, M., Kerwar, S.S., et al., 1994. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature 370, 218±220. Monte®ori, D.C., 1997. Role of complement and Fc receptors in the pathogenesis of HIV-1 infection. Springer Semin. Immunopathol. 18, 371±390. Mullberg, J., Durie, F.H., Otten-Evans, C., Alderson, M.R., RoseJohn, S., Cosman, D., Black, R.A., Mohler, K.M., 1995. A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J. Immunol. 155, 5198±5205. Multani, P.S., Grossbard, M.L., 1999. Monoclonal antibody-based therapies for hematologic malignancies. J. Clin. Oncol. 16, 3691± 3710. Nardin, A., Schlimgen, R., Holers, V.M., Taylor, R.P., 1999. A prototype pathogen bound ex vivo to human erythrocyte complement receptor 1 via bispeci®c monoclonal antibody complexes is cleared to the liver in a mouse model. Eur. J. Immunol. 29, 1581± 1586. Nardin, A., Sutherland, W.M., Hevey, M., Schmaljohn, A., Taylor, R.P., 1998. Quantitative studies of heteropolymer-mediated binding of inactivated Marburg virus to the complement receptor on primate erythrocytes. J. Immunol. Meth. 211, 21±31. Nelson, R.A., 1953. The immune-adherence phenomenon. An immunologically speci®c reaction between microorganisms and erythrocytes leading to enhanced phagocytosis. Science 118, 733±737. Nelson, R.A., 1955. The immune-adherence phenomenon. A hypothetical role of erythrocytes in defense against bacteria and viruses. Proc. Roy. Soc. Med. 49, 55±58. Newman, S.L., Mikus, L.K., Tucci, M.A., 1991. Dierential requirements for the cellular cytoskeleton in human macrophage complement receptor and Fc receptor-mediated phagocytosis. J. Immunol. 146, 967±974. Pascual, M., Danielsson, C., Steiger, G., Schierli, J.A., 1994. Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS. Eur. J. Immunol. 24, 702± 708. Reinagel, M.L., Gezen, M., Ferguson, P.J., Kuhn, S., Martin, E.N., Taylor, R.P., 1997. The primate erythrocyte complement receptor (CR1) as a privileged site. Binding of immunoglobulin G to erythrocyte CR1 does not target erythrocytes for phagocytosis. Blood 89, 1068±1077. Reist, C.J., Combs, M.J., Croft, B.Y., Taylor, R.P., 1993. Antigens pre-bound to the primate erythrocyte complement receptor via cross-linked bispeci®c monoclonal antibody heteropolymers are rapidly cleared from the circulation. Eur. J. Immunol. 23, 3021± 3027. Reist, C.J., Liang, H.Y., Denny, D., Martin, E.N., Scheld, W.M., Taylor, R.P., 1994. Cross-linked bispeci®c monoclonal antibody heteropolymers facilitate the clearance of human IgM from the circulation of squirrel monkeys. Eur. J. Immunol. 24, 2018±2025. Ross, G.D., Yount, W.J., Walport, M.J., Win®eld, J.B., Parker, C.J., Fuller, C.R., et al., 1985a. Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation. J. Immunol. 135, 2005± 2014. Ross, G.D., Cain, J.A., Lachmann, P.J., 1985b. Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin and functions as a receptor for
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835 zymosan and rabbit erythrocytes as well as a receptor for iC3b. J. Immunol. 134, 3307±3315. Schierli, J.A., Taylor, R.P., 1989. Physiological and pathological aspects of circulating immune complexes. Kidney Int. 35, 993± 1003. Schreiber, A.D., Frank, M.M., 1972. Role of antibody and complement in the immune clearance and destruction of erythrocytes I. In vivo eects of IgG and IgM complement ®xing sites. J. Clin. Invest. 51, 575±582. Stahl, P.D., Ezekowitz, R.A.B., 1998. The mannose receptor is a pattern recognition receptor involved in host defense. Curr. Opin. Immunol. 10, 50±55. Tausk, F.A., McCutchan, A., Spechko, P., Schreiber, R.D., Gigli, I., 1986. Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men: varying risk groups for acquired immune de®ciency syndrome. J. Clin. Invest. 78, 977±982. Taylor, R.P., Sutherland, W.M., Martin, E.N., Ferguson, P.J., Reinagel, M.L., Gilbert, E., et al., 1997a. Bispeci®c monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model. J. Immunol. 158, 842±850. Taylor, R.P., Martin, E.N., Reinagel, M.L., Nardin, A., Craig, M., Choice, Q., et al., 1997b. Bispeci®c monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a pro-
835
totype particulate pathogen in a monkey model. J. Immunol. 159, 4035±4044. Taylor, R.P., Ferguson, P.J., Martin, E.N., Cooke, J., Greene, K.L., Grinspun, K., et al., 1997c. Immune complexes bound to primate erythrocyte complement receptor (CR1) via anti-CR1 mAbs are cleared simultaneously with loss of CR1 in a concerted reaction in a rhesus monkey model. Clin. Immunol. Immunopath. 82, 49± 59. Taylor, R.P., Sutherland, W.M., Reist, C.J., Webb, D.J., Wright, E.L., Labuguen, R.H., 1991. Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: a potential therapeutic treatment. Proc. Natl. Acad. Sci. USA 88, 3305±3309. Victoria, J.E., Pierce, W.S., Branks, J.M., Masouredis, S.P., 1990. IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein. J. Lab. Clin. Med. 115, 74±88. Waldmann, T.A., O'Shea, J., 1999. The use of antibodies against the IL-2 receptor in transplantation. Curr. Opin. Immunol. 10, 507± 512. Wright, S.D., 1995. CD14 and innate recognition of bacteria. J. Immunol. 155, 6±8. Wright, S.D., Jong, M.T., 1986. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. J. Exp. Med. 164, 1876±1888.
www.elsevier.com/locate/molimm
How are immune complexes bound to the primate erythrocyte complement receptor transferred to acceptor phagocytic cells? Alessandra Nardin, Margaret A. Lindorfer, Ronald P. Taylor* Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
Abstract Immune complexes (IC) bound to the primate erythrocyte (E) complement receptor (CR1) are cleared from the circulation of primates and localized to phagocytic cells in the liver and spleen without E destruction. IC can be bound to E CR1 either via C3b opsonization or with cross-linked mAb complexes (heteropolymers, HP) which contain a mAb speci®c for CR1 and a mAb speci®c for an antigen. The long-term goal of our work is to apply the HP system to the treatment of human diseases associated with blood-borne pathogens. This review discusses the mechanism by which the E-bound IC are transferred to acceptor cells. Our studies in animal models as well as our in vitro investigations indicate that IC transfer is rapid (usually >90% in 10 min) and does not lead to lysis or phagocytosis of the E. Experiments with speci®c inhibitors and the use of IC prepared with Fab ' fragments suggest that transfer depends mainly upon recognition by Fc receptors on the acceptor cell. Moreover, we ®nd that IC release from the E is associated with a concerted loss of CR1, and is followed by uptake and internalization of the IC by the acceptor cell. We suggest that recognition and binding of the E-bound IC substrates by Fc receptors allows close contact between the E and acceptor cells, which in turn facilitates proteolysis of E CR1, presumably by a macrophage-associated protease. After proteolysis, the released IC are internalized by the macrophages. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Bispeci®c antibodies; Erythrocytes; Immune complexes; Complement receptor 1; Complement
1. Introduction Since the original description of the preparation of monoclonal antibodies (mAbs), much eort has focused on development of mAb-based approaches to treating human diseases (Burnie and Matthews, 1998; Kohler and Milstein, 1975; Multani and Grossbard, 1999; Waldmann and O'Shea, 1999). The long term goal of our work is to develop a general platform immunotherapy for treatment of human diseases associated with blood-borne pathogens (Taylor et al., 1991, 1997a). Fundamental to our approach is the hypothesis that it should be possible to facilitate the safe and rapid clearance of pathogens from the circulation * Corresponding author. Tel.: +1-804-924-2664; fax: +1-804-9245069. E-mail address: [email protected] (R.P. Taylor).
by utilizing and enhancing the paradigm of a natural process, the primate erythrocyte (E) immune complex (IC) clearance reaction (Kuhn et al., 1998; Cornaco et al., 1983; Cosio et al., 1990; Taylor et al., 1991). This reaction is mediated by the E complement receptor (CR1), an intrinsic membrane glycoprotein with speci®city for C3b, a protein fragment generated during complement activation (Fearon, 1980). Complement-opsonized IC containing covalently incorporated C3b form in the circulation and bind to primate E via CR1 in the immune adherence (IA) reaction (Nelson, 1953, 1955). The IC are then safely cleared from the circulation and neutralized in the liver without E destruction (Cornaco et al., 1983; Cosio et al., 1990; Davies, 1996; Davies et al., 1990; Schierli and Taylor, 1989). In numerous in vitro and in vivo models, we have demonstrated that, by making use of bispeci®c mAb
0161-5890/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 9 9 ) 0 0 1 0 3 - 0
828
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
complexes (heteropolymers, HP) which contain a mAb speci®c for CR1 cross-linked with a mAb speci®c for an antigenic site on a target pathogen, we can ensure high anity binding of any target pathogen to E (Nardin et al., 1998; Reist et al., 1993; Taylor et al., 1991, 1997a). The anti-CR1 mAb acts as a surrogate for C3b, and binding is achieved independent of complement opsonization. Moreover, our in vivo experiments in monkeys and in mice have demonstrated that prototype pathogens bound to E CR1 via the HP system are rapidly cleared from the circulation and localized to the liver, thus con®rming our original hypothesis (Nardin et al., 1999; Reist et al., 1993, 1994; Taylor et al., 1997b). Another goal of this work is to obtain a detailed understanding of the basic process by which natural C3b-opsonized and E-bound IC are transferred from E to acceptor phagocytic cells. We believe that the mechanism of this transfer reaction is general and virtually identical for substrates bound to E CR1 via either C3b or HP. The transfer reaction requires the presence of the Fc portions of the antibodies in the IC and results in the concerted loss from E of the CR1 engaged in IC binding. We suggest that close contact between E and acceptor cells, facilitated by Fc receptors, allows one or more membrane associated proteases on the phagocyte to proteolytically cut E CR1 and release it, together with the C3b- or HP-opsonized complex, for subsequent internalization. It is possible that other receptors (e.g., CR3 or CD14 (Wright, 1995; Wright and Jong, 1986)) on the acceptor cell may engage the E-bound IC to allow for the proteolysis and internalization step. However, any release of IC from E in the circulation mediated by Factor I, presumably cleaving C3b to C3bi and then C3dg (Medof et al., 1982), is unlikely to play a signi®cant or direct role in the transfer reaction. We will present evidence to support our hypothesis for the proteolytic mechanism.
Fig. 1. Absence of sequestration of IC-opsonized erythrocytes. 51Crlabeled autologous erythrocytes, with in vitro prebound antibody/125I-dsDNA IC, were infused iv in a chimpanzee and counts were monitored in the circulation. Data are presented as the percentage of counts remaining relative to 1 min values (mean2SD; n = 4). By 10 min, >75% of initially E-bound IC had been ``stripped'' from E, but < 2% of these counts were detectable in the total plasma fraction. By 30 min, 95% of IC had been stripped from E with 06% of counts in the total plasma fraction (1.4% trichloroacetic acid precipitable and 4.5% nonprecipitable). No free 51Cr (<0.2%) was detectable in the plasma samples.
cleared from the circulation without loss of E; >75% of the IC were removed from the E by 10 min and 95% by 30 min (Fig. 1). Our in vitro investigations demonstrated that Factor I mediated-release from E of these complement-opsonized IC is quite slow (Fig. 2)
2. Immune complexes bound to CR1 via C3b or HP can be rapidly and speci®cally cleared from the circulation We have observed that, during in vivo clearance of IC bound to E CR1, the IC themselves, but not the E, are removed from the circulation and localized to the liver. This has held true for IC bound to CR1 either via C3b opsonization or via HP. We performed a series of ex-vivo experiments in which 125I-labeled IC were bound in vitro to 51Cr labeled primate E, reinfused into non-human primates or mice, and then followed in the bloodstream. In experiments in chimpanzees, complement (C3b)-opsonized IgG antidsDNA/125I labeled-dsDNA IC were bound in vitro to autologous 51Cr labeled E and re-infused into the animals (Kimberly et al., 1989). The IC were rapidly
Fig. 2. Release of erythrocyte-bound IC in serum-EDTA. Ab/3Hlabeled dsDNA IC were bound to erythrocytes obtained from several primates (chimpanzee (CH), baboon (BA), rhesus (RH) and cynomolgus (CY) monkeys, human (HU)), and then autologous serum +0.01 M EDTA was added and the percentage of IC released after 1 h was determined. The mean2SD for three independent determinations (with cells and serum from dierent animals) is shown.
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
Fig. 3. Clearance in a rhesus monkey of 125I-labeled IgG antidsDNA antibodies prebound ex vivo via an antigen-based heteropolymer (AHP) to autologous 51Cr-labeled erythrocytes. 51Cr counts in the plasma averaged <2% of erythrocyte-associated counts. The 51 Cr counts after 80 min are approximately twice those for the ®rst 80 min, re¯ecting the cumulative eect of the 2 infusions. Little, if any, 51Cr was cleared. The ®rst line for 125I-IgG (IgG anti-dsDNA from SLE patient Ma) is the theoretical ®t to the data based on double-exponential decay, the second (IgG anti-dsDNA from SLE patient Ha) represent the theoretical ®t to a single exponential decay.
(Edberg et al., 1992). This dierence in release kinetics for the in vivo (fast) vs the in vitro (slow) system indicated that Factor I-mediated degradation of C3b to C3bi and then to C3dg (which does not bind to CR1 (Medof et al., 1982)) is too slow to account for the rapid transfer reaction observed in vivo. Ex-vivo experiments using both HP and antigenbased heteropolymers (AHP) showed a similar pattern of clearance of E-bound IC. In rhesus monkeys, both 125 I labeled proteins prebound to 51Cr labeled E via HP (Reist et al., 1993) and 125I labeled IgG antidsDNA prebound to 51Cr labeled E via an antigenbased HP (anti-CR1 mAb cross linked with dsDNA) (Fig. 3, Ferguson et al., 1995) were cleared from the circulation with similar kinetics (090% in 10 min), and without loss of E. Experiments in A/J mice provided evidence for the generality of the mechanism of the transfer reaction. 125I-labeled bacteriophage FX174 was bound ex-vivo via HP to 51Cr labeled primate E, and, upon infusion into the mouse, >90% of FX174 cleared to the liver in 10 min (Nardin et al., 1999). In both the HP and AHP systems, C3b is not utilized to facilitate E binding. Thus, it is unlikely that factor I (which cleaves C3b but not IgG (Medof et al., 1982)) can play a role in the release of substrates bound to E CR1 via anti-CR1 mAbs in the HP and AHP systems.
829
Fig. 4. IC formed in situ on primate E CR1 are cleared from the circulation coincident with loss of CR1. 125I-labeled anti-CR1 mAb 7G9 (0.8 mg) was infused iv into the circulation of a monkey (5.3 kg), and more than 75% of the infused reagent bound to E. At 61.5 min (see arrow) a bolus of monkey antibodies to mouse IgG was infused. The E pellets were counted to measure bound 125Ilabeled 7G9, and probed with additional 125I-labeled anti-CR1 mAbs 7G9 and HB8592 (initially 2900 and 1140 epitopes per E, respectively) to determine total E CR1. The increase in apparent CR1 epitopes after the infusion at 61.5 min is due to ``capture'' of the mouse mAb probes by the monkey anti-mouse IgG bound to the E. More than 90% of the plasma counts associated with the infused 125Ilabeled mAb 7G9 were also cleared from the circulation by the end of the experiment.
3. In vivo transfer of IC from primate E (or, in nonprimates, platelets) to phagocytic cells involves removal of cell-associated CR1. Historical precedence and recent studies In 1975, Miller et al. reported that platelets of old NZB/NZW mice with chronic IC disease due to a lupus-like syndrome had substantially lost their ability to bind complement-opsonized IC infused into the circulation. In mice and other non-primates IA activity is platelet associated, and it was suggested that ``receptors . . . on the surface membrane of cells from old mice might not be available for binding'' (Miller et al., 1975). This prescient observation is supported by more recent experiments in monkey models and in humans indicating that processing of complement-®xing IC is correlated with a decrease in levels of E CR1, the primate IA receptor (Cosio et al., 1990; Davies, 1996; Davies et al., 1990, 1992). Moreover, E CR1 levels are reduced in individuals with diseases characterized by chronic IC processing such as the auto-immune disease systemic lupus erythematosus (SLE) and AIDS (Fearon, 1985; Iida et al., 1982; Lach-Tri®lie et al., 1999; Ross et al., 1985a; Schierli and Taylor, 1989; Tausk et al., 1986). The parallel between the primate and non-primate systems is therefore clear.
830
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
Fig. 5. Loss of bacteriophage FX174 (black triangles), HP (anti-CR1 mAb 1B4 X anti-FX174 mAb 7B7; black squares) and CR1 (black circles) from biotinylated human E infused iv in the circulation of an A/J mouse as determined by ¯ow cytometry. Approximately 10% of the blood cells were positive for the phycoerythrin-labeled streptavidin used to identify the human E, with virtually no loss of these E from the circulation of the mouse during the experiment. Fluorescein isothiocyanate (FITC)-labeled mAb 7G9, not competing with mAb 1B4 present in the HP, was used to probe for CR1. In a parallel control, the same biotinylated human E (not opsonized with HP and FX174) were infused in another A/J mouse and showed virtually no loss of CR1 (white circles).
We demonstrated loss of E CR1 in a squirrel monkey in a study of HP-mediated clearance of human IgM, which was selected as a prototype pathogen (Reist et al., 1994). First human IgM, then HP (anti-CR1 mAb cross-linked with anti-human IgM mAb) were infused into the circulation of the monkey. Separate radiolabels on the HP and IgM allowed us to demonstrate that the HP facilitated binding of human IgM to the monkey E. Soon after HP infusion and binding of the substrate to E, both HP and IgM were cleared from the circulation and localized to the liver. Moreover, coincident with clearance of these E-bound substrates, CR1 levels on the monkey E also decreased considerably (Reist et al., 1994). We sought to generalize the clearance reaction by selecting a simpler model: IC were formed directly on E CR1 by infusing ®rst large amounts of 125I-labeled anti-CR1 mouse mAb 7G9, then monkey anti-mouse IgG (Fig. 4) (Taylor et al., 1997c). The IC does indeed form on the E, and it is cleared together with CR1 in a concerted reaction. As mentioned above, in a mouse model we found that the bacteriophage FX174 is cleared to the liver if it is infused bound to primate E via HP. Flow cytometric analyses of this reaction demonstrate simultaneous loss of FX174, HP, and CR1 from the population of
human E infused into the mouse (Fig. 5) (Nardin et al., 1999). Independent studies by Ulevitch and Frank demonstrated that infusion of LPS into the bloodstream of rabbits or guinea pigs leads to a rapid but transient loss of platelets from the bloodstream, in a reaction which requires complement activation (Kane et al., 1973; Mathison and Ulevitch, 1979, 1981). They reported that, immediately after infusion, a large fraction of the LPS is bound to platelets. Furthermore, the majority of the LPS is rapidly cleared from the circulation and localized to the liver. It is likely that IA of C3b-opsonized LPS/IgG IC to platelets (mediated by endogenous antibodies and complement) causes the platelets to bind transiently to Fc receptors on ®xed tissue macrophages in the liver. Proteolysis and removal of the platelets' IA receptors containing bound IC would allow these cells to be released back into the bloodstream. It is important to note that the platelets which reappear in the circulation no longer bind LPS, although they do bind LPS after it is ®rst infused (Kane et al., 1973; Mathison and Ulevitch, 1979, 1981). These platelets should have severely reduced IA activity if tested with complement-opsonized substrates, compared to platelets isolated from the animals before the experiment. Removal of the IA receptor from non-primates' platelets or of CR1 from primate E appears to be a necessary condition for transfer of cell-bound IC to phagocytic cells in the liver. We suggest that close association of the E-bound IC with ®xed tissue macrophages (mediated by Fc and possibly other receptors) is followed by a proteolytic cut of CR1. Phagocytic cells express numerous membrane associated proteases (Bauvois et al., 1992), and E CR1 is quite sensitive to proteolysis and can easily be removed from E by a variety of proteases (Pascual et al., 1994). Several membrane-associated proteins, including CR2, L-selectin, IL-6R, TNF-aR, and CD16, can be shed from cells by protease activity. In some cases, a Zn-dependent metalloprotease, not susceptible to common protease inhibitors, has been found responsible for the proteolytic cleavage (Chen et al., 1995; FremeauxBacchi et al., 1996; Huizinga et al., 1990; Mohler et al., 1994; Mullberg et al., 1995). In fact, L-selectin and CR2 each contain multiple short consensus repeat motifs (Ahearn and Fearon, 1989) similar to those expressed on CR1, and are released from leukocytes and lymphocytes, respectively, by cleavage in a domain proximal to the transmembrane portion. Recently Hamer et al. (1998) have studied COS cells transfected with membrane-bound CR1. Their work suggests that CR1 can be released after a proteolytic cut within the transmembrane region. In these reactions a protein is ``shed'' from the cell due to a protease present on the cell itself. However, in the case of E CR1, it is likely
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
Fig. 6. Infused Fab' X Fab' HP does not mediate clearance unless followed by intact IgG anti-FX174 mAb. At time zero, 100 mg of 131 I-labeled bacteriophage FX174 was infused into the circulation of a 8.7 kg monkey, and the Fab' X Fab' HP was infused 48 min later. Although a very high level of E binding accompanied infusion of the HP, E-bound counts did not clear from the circulation, and liver counts remained ¯at. Forty min later (see arrow on the x axis), whole anti-FX174 mAb 7B7 was infused, and, after a 5-min delay, clearance commenced and proceeded rapidly. Anger camera was used for imaging: background (before infusion) was 2000 counts, liver background was 130 counts, and a total of 31,000 counts were infused.
that the protease(s) which causes release of E CR1 is provided by the phagocytic cell, and not by the E. We propose that a Zn-dependent metalloprotease provided by the acceptor cell can cause release of E CR1, and our preliminary studies (Craig and Taylor, manuscript in preparation) support this mechanism. An extensive literature documents the importance of anti-E antibodies in autoimmune hemolytic anemias (Schreiber and Frank, 1972; Victoria et al., 1990). Opsonization of E with antibodies against surface epitopes has traditionally been used to generate model substrates for in vitro study of phagocytosis and for in vivo investigation of the mechanisms by which antibody-opsonized particles are removed from the bloodstream (Edberg and Kimberly, 1992; Frank, 1989; Schreiber and Frank, 1972). In view of these facts, it is surprising that the primate E can serve as an IC carrier without itself being destroyed. We believe that E CR1 presents a possibly unique and privileged site on the E surface. Complement-opsonized IC containing IgG can indeed bind naturally to E via CR1 without causing E destruction. We have demonstrated that binding of large amounts of IgG to CR1 on primate E does not lead to phagocytosis of the E by monocytes (Reinagel et al., 1997). Instead, when IgG is bound to other sites on E, the cells are indeed phagocytosed. These disparate observations are reconciled by allowing for pro-
831
Fig. 7. Clearance in A/J mice of 125I-labeled bacteriophage FX174 bound ex vivo to 51Cr-labeled human E via Fab' HP and then infused iv into the circulation of the mouse (black squares). Data are presented as the percentage of 125I counts remaining bound to the 51 Cr-labeled E. In one case FX174 was bound to the E and then further opsonized with anti-FX174 mAb 7B7 before injection into the mouse (black circles).
teolysis of CR1. If all E-associated IgG is bound to CR1, which is then released by proteolytic cleavage from the E, naturally the E will be spared, as the opsonizing reagent (IgG) will be removed. This mechanism should work equally well if IC are bound to CR1 via C3b, the natural ligand, or via the anti-CR1 mAb in the HP.
4. Intact mAbs are required for E CR1 bound IC recognition and transfer to phagocytic cells. The role of Fc and other receptors Both Fc receptors and complement are likely to participate in the transfer reaction. In applying the HP system in both monkey and mouse models, we found that the presence of intact IgG on the E-bound IC was an absolute requirement for ecient and rapid transfer. In several experiments we observed rapid E binding and clearance of bacteriophage FX174 through an anti-FX174 X anti-CR1 HP. However, Fig. 6 shows that an Fab ' X Fab ' HP administered to a monkey after infusion of 131I labeled FX174 was sucient to ensure quantitative binding of bacteriophage to E but unable to facilitate clearance (Taylor et al., 1997b). Only after an intact anti-FX174 mAb was injected into the circulation (and bound to other sites on the Ebound substrate) were counts cleared from the E and localized to the liver. Similarly, in mice, clearance of
832
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
125
I labeled FX174 bound to E (human or monkey) via an Fab ' based HP was slow. Additional binding of an intact anti-FX174 mAb to the E-Fab ' HP-FX174 complex restored the normal clearance pattern (Fig. 7) (Nardin et al., 1999). The need for the Fc portion of the mAbs could re¯ect engagement of Fc receptors as well as a requirement for complement activation and opsonization of the IC, with subsequent involvement of complement receptors such as CR1 or CR3 on the phagocytes (Ahearn and Fearon, 1989; Hermanowski-Vosatka et al., 1988; Ross et al., 1985b). In the mouse model, interference with complement activation at the C3b opsonization step, through use of either cobra venom factor or mouse complement regulatory protein Crry, resulted in impaired clearance of E-bound FX174 (Nardin et al., 1999). However, in vitro studies of transfer of HP-bound FX174 from E to the murine macrophage cell line P388D1 showed that ecient transfer occurred in the absence of complement (Reinagel and Taylor, submitted for publication). An in vitro system which focused on transfer of E. coli, bound to E via HP, to acceptor monocytes provides further insight (Kuhn et al., 1998). Blockade of actin polymerization with cytochalasin D inhibited internalization, but use of colchicine (which blocks microtubule formation) did not inhibit transfer. Since both FcR and CR1 require intact actin ®laments for phagocytosis (Greenberg and Silverstein, 1993; Newman et al., 1991) but CR1 also requires microtubule assembly, internalization in our in vitro model is apparently FcR mediated. Other receptors may play a role in transfer, depending upon the nature of the substrate bound to E CR1. For example, CD14, CR3, and the mannose receptor can bind LPS and other bacterial structures (Stahl and Ezekowitz, 1998; Wright, 1995; Wright and Jong, 1986): interaction of these receptors with bacteria bound to E via HP could allow for close juxtaposition of the respective cells, followed by proteolysis of CR1 and release of bacteria. In fact, we found that blockade of Fc receptors alone only weakly blocked monocyte mediated release from E of HP-bound E. coli (Kuhn et al., 1998). However, uptake of the E. coli by monocytes was inhibited more eectively. Thus, close and sustained contact between E and phagocyte, even if mediated by receptors unable to support internalization, could be sucient for cleavage of CR1 and removal of the CR1-HP-Ag complex from E. CR3 binds LPS in an EDTA-inhibitable fashion and independent from C3bi opsonization (Wright, 1995; Wright and Jong, 1986). Although EDTA signi®cantly blocked uptake of E. coli by the monocytes, it seems unlikely that CR3 alone was responsible for the complete transfer reaction, since in the absence of HP the bacteria were weakly taken up by the monocytes.
Fig. 8. Clearance from the circulation of monkeys of 125I-labeled E. coli prebound ex vivo to autologous 51Cr-labeled E via either C3b opsonization (autologous serum was used) or an HP made with antiCR1 mAb 7G9 cross linked with anti-E. coli mAb 44. Monkey A (rhesus) was infused ®rst with E-HP-E. coli, then, at 51 min, with EC3b-E. coli. Monkey B (stump-tailed macaque) was infused ®rst with E-C3b-E. coli, then, at 51 min, with E-HP-E. coli; for the experiment in monkey B, 2 times more 125I-labeled E. coli were bound to 5 times less 51Cr-labeled E.
5. Direct comparisons of the transfer reaction for IC bound to E via C3b or via the HP (or AHP) systems The key presumption in our approach is that, with respect to the transfer reaction, IC bound to E via anti-CR1 mAbs (HP and AHP) will be processed in the same fashion as natural C3b-opsonized IC bound to E. Our recent work in an in vitro model, on the prototypical IC dsDNA/IgG anti-dsDNA, con®rms this hypothesis. Since our studies on this IC system 10 years ago (Kimberly et al., 1989) ®rst lead to our working hypothesis on the mechanism of the transfer reaction and to the development of HP and AHP, it is reassuring that the present studies continue to be consistent with the original postulate. We used the same SLE IgG anti-dsDNA antibodies to prepare both types of E-associated IC, and then studied transfer to U937 cells. Both C3b-opsonized dsDNA/anti-dsDNA IC and the AHP-anti-dsDNA IC, after ®rst binding to E, are transferred to U937 cells with similar and rapid kinetics. Moreover, monomeric mouse IgG2a, which
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
binds well to the high avidity Fc receptor, inhibits transfer of both types of IC, and transfer of the AHP complex is accompanied by loss of E CR1 (Craig and Taylor, abstract submitted). We performed an ex-vivo study to examine clearance of E. coli in monkeys after it is bound to E. In this experiment 125I-labeled E. coli was bound in vitro to 51 Cr-labeled monkey E, either via complement opsonization with autologous monkey serum or with a speci®c HP (Kuhn et al., 1998). The doubly-labeled substrates were then consecutively infused into the circulation of two monkeys, and the rate of clearance of infused materials was measured. Fig. 8 indicates that the clearance kinetics for the two substrates are quite similar. E-bound 125I-labeled E. coli is cleared quickly in both cases, with >95% clearance in less than 10 min. There is no loss of the E that had bound C3bopsonized bacteria, although there is a small but transient loss from the circulation of the E containing HP and E. coli. This experiment, with a dierent substrate from the antibody/dsDNA IC, provides further evidence that IC bound to E are handled in a similar fashion, whether they are bound via C3b opsonization or via a HP construct in which the mAb to CR1 serves as a surrogate for C3b. This precludes a role for factor I in the transfer reaction and is in agreement with earlier in vitro investigations in other model systems (Emlen et al., 1989, 1992). 6. Future considerations Several infectious agents are known to express ligands speci®c for receptors present on cells belonging to the reticuloendothelial system (RES), so that entrance results in productive infection instead of destruction of the pathogen (e.g., HIV and Marburg virus (Becker et al., 1995; Fauci, 1996; Monte®ori, 1997)). However, there are precedents for the use of bispeci®c antibody complexes in redirecting the pathogen through Fc receptors into a cell compartment where it can be eciently killed (Connor et al., 1991). One of the major challenges for our future work will be to determine if use of the HP-E system will allow for presentation of bona®de pathogens to the RES under conditions which do indeed lead to neutralization and destruction of the pathogens. Acknowledgements This work was supported by the Defense Advanced Research Projects Agency, Order MDA972-96-K-001 under contract MDA972-96-K-003, and by NIH grant AR43307. The authors would like to thank Dr P. Klebba for kindly providing the E. coli and speci®c
833
mAb, and Drs W. Emlen and J. Edberg for stimulating discussions. Figs. 1±7 were all reproduced with permission from the respective journals. Fig. 1: The Journal of Clinical Investigation (Kimberly et al., 1989). Figs. 2, 5 and 7: The European Journal of Immunology (Edberg et al., 1992; Nardin et al., 1999). Fig. 3: Arthritis and Rheumatism (Ferguson et al., 1995). Fig. 4: Clinical Immunology and Immunopathology (Taylor et al., 1997c). Fig. 6: The Journal of Immunology (Taylor et al., 1997b. Copyright 1997. The American Association of Immunologists).
References Ahearn, J.M., Fearon, D., 1989. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46, 183±219. Bauvois, B., Sanceau, J., Wietzerbin, J., 1992. Human U937 cell surface peptidase activities: characterization and degradative eect on tumor necrosis factor. Eur. J. Immunol. 22, 923±930. Becker, S., Spiess, M., Klenk, H., 1995. The asialoglycoprotein receptor is a potential liver-speci®c receptor for Marburg virus. J. Gen. Virol. 76, 393±399. Burnie, J.P., Matthews, R.C., 1998. The renaissance of antibody therapy. J. Antimicrob. Chemother. 41, 319±322. Chen, A., Engel, P., Tedder, T.F., 1995. Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. J. Exp. Med. 183, 519±530. Connor, R.I., Dinces, N.B., Howell, A.L., Romet-Lemonne, J.L., Pasquali, J.L., Fanger, M.W., 1991. Receptors for IgG on human monocytes and macrophages are not infectivity receptors for human immunode®ciency virus type 1: studies using bispeci®c antibodies to target HIV-1 to various myeloid cell surface molecules including the FcR. Proc. Natl. Acad. Sci. USA 88, 9593± 9597. Cornaco, J.B., Hebert, L.A., Smead, W.L., Van Aman, M.E., Birmingham, D.J., Waxman, F.J., 1983. Primate erythrocyteimmune complex-clearing mechanism. J. Clin. Invest. 71, 236± 247. Cosio, F.G., Xiao-Ping, S., Birmingham, D.J., Van Aman, M., Hebert, L.A., 1990. Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates. J. Immunol. 145, 4198±4206. Davies, K.A., 1996. Complement, immune complexes and systemic lupus erythematosus. Brit. J. Rheum. 35, 5±23. Davies, K.A., Hird, V., Stewart, S., Sivolapenko, G.B., Jose, P., Epenetos, A.A., et al., 1990. A study of in vivo immune complex formation and clearance in man. J. Immunol. 144, 4613±4620. Davies, K.A., Peters, M., Beynon, H.L., Walport, M.J., 1992. Immune complex processing in patients with systemic lupus erythematosus. J. Clin. Invest. 90, 2075±2083. Edberg, J.C., Kimberly, R.P., Taylor, R.P., 1992. Functional characterization of non-human primate erythrocyte immune adherence receptors: implications for the uptake of immune complexes by the cells of the mononuclear phagocytic system. Eur. J. Immunol. 22, 1333±1339. Edberg, J.C., Kimberly, R.P., 1992. Receptor speci®c probes for the study of Fc receptor speci®c function. J. Immunol. Meth. 148, 179±187. Emlen, W., Burdick, G., Carl, V., Lachmann, P.J., 1989. Binding of
834
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835
model immune complexes to erythrocyte CR1 facilitates immune complex uptake by U937 cells. J. Immunol. 142, 4366±4371. Emlen, W., Carl, V., Burdick, G., 1992. Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes. Clin. Exp. Immunol. 89, 8±17. Fauci, A.S., 1996. Host factors and the pathogenesis of HIV-induced disease. Nature 384, 529±534. Fearon, D.T., 1980. Identi®cation of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152, 20±30. Fearon, D.T., 1985. Human complement receptors for C3b (CR1) and C3 d (CR2). J. Invest. Dermatol. 85, 53±57. Ferguson, P.J., Reist, C.J., Martin, E.N., Johnson, C., Greene, K.L., Kuhn, S., et al., 1995. Antigen-based heteropolymers: a potential therapy for binding and clearing autoantibodies via erythrocyte CR1. Arth. Rheumat. 38, 190±200. Frank, M.M., 1989. The role of macrophages in bloodstream clearance. In: Human Monocytes. Academic Press, New York, pp. 337±344. Fremeaux-Bacchi, V., Bernard, I., Maillet, F., Mani, J., Fontaine, M., Bonnefoy, J., et al., 1996. Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-barr virus receptor, CR2) that binds iC3b and CD23. Eur. J. Immunol. 26, 1497±1503. Greenberg, S.C.P., Silverstein, S.C., 1993. Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 177, 529±534. Hamer, I., Paccaud, J.-P., Belin, D., Maeder, C., Carpentier, J.-L., 1998. Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain. Biochem. J. 329, 183±190. Hermanowski-Vosatka, A., Detemers, P.A., Gotze, O., Silverstein, S.C., Wright, S.D., 1988. Clustering of ligand on the surface of a particle enhances adhesion to receptor-bearing cells. J. Biol. Chem. 263, 17,822±17,827. Huizinga, T.W.J., de Haas, M., Kleijer, M., Nuijens, J.H., Roos, D., von dem Borne, A.E.G.Kr, 1990. Soluble Fc receptor III in human plasma originates from release by neutrophils. J. Clin. Invest. 86, 416±423. Iida, K., Mornaghi, R., Nussenzweig, V., 1982. Complement receptor (CR1) de®ciency in erythrocytes from patients with systemic lupus erythematosus. J. Exp. Med. 155, 1427±1438. Kane, M.A., May, J.E., Frank, M.M., 1973. Interactions of the classical and alternative complement pathway with endotoxin lipopolysaccharide. J. Clin. Invest. 52, 370±376. Kimberly, R.P., Edberg, J.C., Merriam, L.T., Clarkson, S.B., Unkeless, J.C., Taylor, R.P., 1989. In vivo handling of soluble complement ®xing Ab/dsDNA immune complexes in chimpanzees. J. Clin. Invest. 84, 962±970. Kohler, G., Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of prede®ned speci®city. Nature 256, 495±497. Kuhn, S.E., Nardin, A., Klebba, P.E., Taylor, R.P., 1998. Escherichia coli bound to the primate erythrocyte complement receptor via bispeci®c monoclonal antibodies are transferred to and phagocytosed by human monocytes in an in vitro model. J. Immunol. 160, 5088±5097. Lach-Tri®lie, E., Marfurt, J., Schwarz, S., Sadallah, S., Schierli, J.A., 1999. Complement receptor 1 (CD35) on human reticulocytes: normal expression in systemic lupus erythematosus and HIV-infected patients. J. Immunol. 162, 7549±7554. Mathison, J.C., Ulevitch, R.J., 1979. The clearance, tissue distribution, and cellular localization of intravenously injected lipopolysaccharide in rabbits. J. Immunol. 123, 2133±2143. Mathison, J.C., Ulevitch, R.J., 1981. In vivo interaction of bacterial lipopolysaccharide (LPS) with rabbit platelets: modulation by C3 and high density lipoproteins. J. Immunol. 126, 1575±1580. Medof, M.E., Prince, G.M., Oger, J.J.F., 1982. Kinetics of inter-
action of immune complexes with complement receptors on human red blood cells: modi®cation of complexes during interaction with red cells. Clin. Exp. Immunol. 48, 715±725. Miller, G.W., Steinberg, A.D., Green, I., Nussenzweig, V., 1975. Complement-dependent alterations in the handling of immune complexes by NZB/W mice. J. Immunol. 114, 1166±1170. Mohler, K.M., Sleath, P.R., Fitzner, J.N., Cerretti, D.P., Alderson, M., Kerwar, S.S., et al., 1994. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature 370, 218±220. Monte®ori, D.C., 1997. Role of complement and Fc receptors in the pathogenesis of HIV-1 infection. Springer Semin. Immunopathol. 18, 371±390. Mullberg, J., Durie, F.H., Otten-Evans, C., Alderson, M.R., RoseJohn, S., Cosman, D., Black, R.A., Mohler, K.M., 1995. A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J. Immunol. 155, 5198±5205. Multani, P.S., Grossbard, M.L., 1999. Monoclonal antibody-based therapies for hematologic malignancies. J. Clin. Oncol. 16, 3691± 3710. Nardin, A., Schlimgen, R., Holers, V.M., Taylor, R.P., 1999. A prototype pathogen bound ex vivo to human erythrocyte complement receptor 1 via bispeci®c monoclonal antibody complexes is cleared to the liver in a mouse model. Eur. J. Immunol. 29, 1581± 1586. Nardin, A., Sutherland, W.M., Hevey, M., Schmaljohn, A., Taylor, R.P., 1998. Quantitative studies of heteropolymer-mediated binding of inactivated Marburg virus to the complement receptor on primate erythrocytes. J. Immunol. Meth. 211, 21±31. Nelson, R.A., 1953. The immune-adherence phenomenon. An immunologically speci®c reaction between microorganisms and erythrocytes leading to enhanced phagocytosis. Science 118, 733±737. Nelson, R.A., 1955. The immune-adherence phenomenon. A hypothetical role of erythrocytes in defense against bacteria and viruses. Proc. Roy. Soc. Med. 49, 55±58. Newman, S.L., Mikus, L.K., Tucci, M.A., 1991. Dierential requirements for the cellular cytoskeleton in human macrophage complement receptor and Fc receptor-mediated phagocytosis. J. Immunol. 146, 967±974. Pascual, M., Danielsson, C., Steiger, G., Schierli, J.A., 1994. Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS. Eur. J. Immunol. 24, 702± 708. Reinagel, M.L., Gezen, M., Ferguson, P.J., Kuhn, S., Martin, E.N., Taylor, R.P., 1997. The primate erythrocyte complement receptor (CR1) as a privileged site. Binding of immunoglobulin G to erythrocyte CR1 does not target erythrocytes for phagocytosis. Blood 89, 1068±1077. Reist, C.J., Combs, M.J., Croft, B.Y., Taylor, R.P., 1993. Antigens pre-bound to the primate erythrocyte complement receptor via cross-linked bispeci®c monoclonal antibody heteropolymers are rapidly cleared from the circulation. Eur. J. Immunol. 23, 3021± 3027. Reist, C.J., Liang, H.Y., Denny, D., Martin, E.N., Scheld, W.M., Taylor, R.P., 1994. Cross-linked bispeci®c monoclonal antibody heteropolymers facilitate the clearance of human IgM from the circulation of squirrel monkeys. Eur. J. Immunol. 24, 2018±2025. Ross, G.D., Yount, W.J., Walport, M.J., Win®eld, J.B., Parker, C.J., Fuller, C.R., et al., 1985a. Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation. J. Immunol. 135, 2005± 2014. Ross, G.D., Cain, J.A., Lachmann, P.J., 1985b. Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin and functions as a receptor for
A. Nardin et al. / Molecular Immunology 36 (1999) 827±835 zymosan and rabbit erythrocytes as well as a receptor for iC3b. J. Immunol. 134, 3307±3315. Schierli, J.A., Taylor, R.P., 1989. Physiological and pathological aspects of circulating immune complexes. Kidney Int. 35, 993± 1003. Schreiber, A.D., Frank, M.M., 1972. Role of antibody and complement in the immune clearance and destruction of erythrocytes I. In vivo eects of IgG and IgM complement ®xing sites. J. Clin. Invest. 51, 575±582. Stahl, P.D., Ezekowitz, R.A.B., 1998. The mannose receptor is a pattern recognition receptor involved in host defense. Curr. Opin. Immunol. 10, 50±55. Tausk, F.A., McCutchan, A., Spechko, P., Schreiber, R.D., Gigli, I., 1986. Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men: varying risk groups for acquired immune de®ciency syndrome. J. Clin. Invest. 78, 977±982. Taylor, R.P., Sutherland, W.M., Martin, E.N., Ferguson, P.J., Reinagel, M.L., Gilbert, E., et al., 1997a. Bispeci®c monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model. J. Immunol. 158, 842±850. Taylor, R.P., Martin, E.N., Reinagel, M.L., Nardin, A., Craig, M., Choice, Q., et al., 1997b. Bispeci®c monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a pro-
835
totype particulate pathogen in a monkey model. J. Immunol. 159, 4035±4044. Taylor, R.P., Ferguson, P.J., Martin, E.N., Cooke, J., Greene, K.L., Grinspun, K., et al., 1997c. Immune complexes bound to primate erythrocyte complement receptor (CR1) via anti-CR1 mAbs are cleared simultaneously with loss of CR1 in a concerted reaction in a rhesus monkey model. Clin. Immunol. Immunopath. 82, 49± 59. Taylor, R.P., Sutherland, W.M., Reist, C.J., Webb, D.J., Wright, E.L., Labuguen, R.H., 1991. Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: a potential therapeutic treatment. Proc. Natl. Acad. Sci. USA 88, 3305±3309. Victoria, J.E., Pierce, W.S., Branks, J.M., Masouredis, S.P., 1990. IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein. J. Lab. Clin. Med. 115, 74±88. Waldmann, T.A., O'Shea, J., 1999. The use of antibodies against the IL-2 receptor in transplantation. Curr. Opin. Immunol. 10, 507± 512. Wright, S.D., 1995. CD14 and innate recognition of bacteria. J. Immunol. 155, 6±8. Wright, S.D., Jong, M.T., 1986. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. J. Exp. Med. 164, 1876±1888.