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The Journal of Immunology, 2000, 164: 1977-1985.
Copyright © 2000 by The American Association of Immunologists

Transfer of Immune Complexes from Erythrocyte CR1 to Mouse Macrophages1

Michele L. Reinagel and Ronald P. Taylor2

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are developing a potential therapeutic approach for removing pathogens from the circulation of primates in which the pathogen is bound to the complement receptor (CR1) on E using a bispecific mAb complex, a heteropolymer (HP). We have used mAb this approach to demonstrate that cleared prototype pathogens are localized to, phagocytosed in, and destroyed in the liver. Extension of this work to a clinical setting will require a detailed understanding of the mechanism by which the E-bound immune complex substrates are transferred to fixed tissue macrophages in the liver, the transfer reaction. Therefore, we examined an in vitro system to study this process using bacteriophage {phi}X174 as a model pathogen. E containing {phi}X174 (bound via an anti-CR1/anti-{phi}X174 HP) were incubated with P388D1 murine macrophages, and the two cell types were separated by centrifugation through Ficoll. Both E and macrophages were then probed and analyzed by RIA or flow cytometry. The results indicate that all three components of the E-bound IC ({phi}X174, HP, and CR1) were removed from the E and internalized by the macrophages. We found that transfer requires the Fc portion of IgG, because little transfer of {phi}X174 occurs when it is bound to E CR1 using a HP containing only Fab fragments. These findings, taken in the context of other studies, suggest a general mechanism for the transfer reaction in which Fc receptors facilitate close juxtaposition of the macrophage to the E-bound IC which then allows a macrophage-associated protease to cleave CR1. The released IC are then internalized and processed by the macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In 1953 Nelson (1) performed a series of in vitro experiments that demonstrated that complement-opsonized Ab/bacteria immune complexes (ICs)3 bound to E via immune adherence and were then phagocytosed by leukocytes. This work suggested that primate E might play an important role in defense against certain infectious diseases. Subsequent investigations have revealed many of the most important details of the immune adherence reaction. A variety of ICs, including those formed between soluble proteins and specific IgG Abs, can activate complement, covalently capture C3b, and then bind to E via complement receptor 1 (CR1) (2, 3, 4). In fact, in vivo studies have demonstrated that C3b-opsonized IC which bind to E in the circulation are subsequently safely cleared from the bloodstream and phagocytosed in the liver and spleen without E destruction (3, 5, 6). Although not explicitly demonstrated, the clearance reaction is presumed to be facilitated by fixed tissue macrophages (e.g., liver Kupffer cells) (7, 8).

Use of appropriate specific mAbs to promote immune adherence of pathogens to primate E would appear to be a reasonable way of developing an immunological defense against pathogens in the bloodstream. However, the degree of binding of complement-opsonized IC to primate E can be highly variable (~20% to >95%), depending on the efficiency of capture of C3b by the IC (2, 5, 9, 10, 11). To ensure a high level of binding (>90%) of virtually any target substrate to E and to develop a potential therapy for infectious diseases, we have produced bispecific mAb complexes (heteropolymer(s), HP) consisting of a mAb to CR1 that is chemically cross-linked to a mAb specific for the target pathogen (12, 13, 14). The fundamental presumption in this approach is that the anti-CR1 mAb in the HP will act as a surrogate for C3b; thus, the pathogen should be bound to E via the HP and then be cleared from the circulation by the same mechanism that operates during clearance of complement-opsonized and E-bound IC. We have demonstrated that specific HP promote a high level of binding of a variety of substrates to primate E. In addition, in vivo studies in monkey models indicate that prototype pathogens (such as the bacteriophage {phi}X174) bound to E via the HP construct are also cleared from the circulation and destroyed in the liver, without loss of E (15, 16). However, the details of the mechanism by which these E-bound IC are cleared from the circulation and transferred to fixed tissue macrophages remain to be delineated (17).

Therefore, we are developing in vitro systems designed to model the in vivo IC transfer reaction. In the present studies, we used a mouse macrophage tumor cell line (P388D1) as the acceptor cell and E-bound IC prepared with primate E, {phi}X174, and specific HP. Use of the mouse cell line eliminates the potential complication of location of primate CR1 on the surface of both the phagocytic cell and E. Through the use of this in vitro model, we can demonstrate a loss of {phi}X174, HP, and CR1 from the E (in the absence of complement) and a gain of all three components by the phagocytic cell, suggesting that CR1 is removed from the E as a key step in the transfer reaction. Our findings also indicate that transfer of IC from E to macrophages requires interaction of the Fc portion of IgG (contained within the IC) with one or more Fc receptors on the macrophage surface. In addition, we show that inhibition of the cytoskeletal rearrangement of the macrophage by blocking actin polymerization partially blocks IC transfer. Finally, we observe a similar transfer reaction in experiments in which a human cell line (U937) (18) is used as the acceptor cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MAbs, HP, and other reagents

Purified anti-CR1 mouse mAbs 7G9 (IgG2a), HB8592, and 1B4 (both IgG1) have been described (19, 20, 21, 22). Bacteriophage {phi}X174 was kindly provided by Professor Nino Incardona, and mAbs specific for {phi}X174 (7B7 and {phi}X51.1) have also been reported (16). HP were prepared by covalently cross-linking mAb 7G9 or HB8592 with anti-{phi}X174 mAb 7B7 (IgG2a) (anti-CR1 x anti-{phi}X174: 7G9 x 7B7 and HB8592 x 7B7) via a thioether bond as described (16, 23). Fab anti-CR1 (7G9) x Fab anti-{phi}X174 (7B7) HP was constructed by Medarex (Annandale, NJ). MAb 2.4G2, specific for murine Fc{gamma}RII/Fc{gamma}RIII (24), was kindly provided by Professor J. Edberg. Fluorescein-labeled neutralite avidin (FITC-NA) and NA conjugated to HRP (NA-HRP) were purchased from Southern Biotechnology Associates (Birmingham, AL). RPMI 1640, FCS, and penicillin/streptomycin were purchased from Life Technologies (Gaithersburg, MD). Cytochalasin D, Triton X-100 (TX100), o-phenylenediamine, and a general protease inhibitor (P-2714) were purchased from Sigma (St. Louis, MO). Several mAbs and polyclonal Abs as well as {phi}X174 were labeled with 125I or were covalently conjugated with biotin or FITC as described previously (25, 26, 27).

Cells

E and serum were obtained with informed consent from a variety of human donors, and in one experiment (as noted below) E were also obtained from a cynomolgus monkey. E were stored as 25% dispersions in Alsever’s solution. For several experiments, E were 51Cr-labeled with an average of 2500 cpm per 1 x 106 cells (28). The P388D1 murine macrophage cell line (TIB 63) and the U937 human monocytic cell line (CRL 1593) were purchased from American Type Culture Collection (Manassas, VA). P388D1 cells were cultured as adherent monolayers and U937 cells were cultured in suspension in RPMI 1640 supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin and were split just before confluency. P388D1 cells were resuspended by vigorous pipetting using cold RPMI media. Preliminary experiments (not shown) confirmed that there was no specific binding of the mouse anti- human CR1 mAbs to the P388D1 cells.

Binding of ICs to E

E were washed three times with PBS containing 1% BSA (BSA-PBS) and reconstituted to suspensions of 10–40%. HP and {phi}X174 were bound to E by one of two methods. In the first protocol, {phi}X174 was first added to the E, and after mixing, HP (7G9 x 7B7 or HB8592 x 7B7) was added. The samples were incubated at 37°C for 15 min, washed three times, and reconstituted to the initial E concentration. Alternatively, HP were added to the E suspension, and after a 15-min incubation the E were washed three times, reconstituted, and then mixed with {phi}X174 followed by an incubation and three washes. During several experiments, a large quantity of E were opsonized in bulk with HP and {phi}X174 and split into individual samples to minimize differences between samples. Depending on the design of the experiment, the E were labeled with 51Cr and/or the {phi}X174 was labeled with 125I, biotin, or FITC. C3b deposition on E containing bound HP-{phi}X174 IC was accomplished by incubating the cells in 40% normal human serum for 15 min at 37°C (29).

IC transfer reaction

E containing bound IC were mixed with P388D1 cells or U937 cells (in RPMI-FCS) at various cellular ratios. The mixed cells were pelleted by centrifugation at 1258 x g for 5 min and were then incubated for between 5 min and 2 h at 37°C. Macrophages were then separated from the E by layering the resuspended cell mixture over Ficoll-Paque Plus (P388D1) or 60% Percoll in PBS (both reagents Pharmacia, Piscataway, NJ) (U937) and centrifuging for 15 min at 1258 x g. After a wash with BSA-PBS, the separated cells were incubated for 15 min at 37°C with a labeled Ab or with FITC-NA. During certain experiments, the macrophages were fixed with 1% paraformaldehyde posttransfer and then permeabilized for 40 min at room temperature using 0.5% TX100 before the addition of labeled Ab.

Cells were analyzed for loss or gain of IC by RIA or by flow cytometry on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA). FCSC beads (Flow Cytometry Standards, San Juan, PR) were used to normalize the log fluorescence (measured by flow cytometry) to mean equivalent soluble fluorochromes (MESF). In preliminary experiments, the FCS was first heat inactivated (to destroy complement) before it was added to the RPMI. This procedure had no effect on the transfer reaction (not shown).

Demonstration of macrophage-associated CR1 by ELISA

Transfer experiments similar to those described above were performed with the following modifications. Approximately 2 x 107 P388D1 cells were mixed with 2 x 108 E-HP-{phi}X174 IC or naive E. The cell mixtures were pelleted and incubated for 10–40 min at 37°C. After separation, residual E were lysed to ensure complete removal of the E from the macrophages (30): 1.5 ml phosphate-buffered 0.1% NaCl was added to the macrophages, and after 40 s 1.5 ml phosphate-buffered 1.8% NaCl was added to reequilibrate the cellular suspension. After centrifugation and one wash with BSA-PBS, the macrophages were lysed using 0.5% TX100 for 40 min at room temperature in the presence of a general protease inhibitor mixture. Next, 100 µl of the lysate were placed into each of four wells of a tissue culture-treated 96-well flat-bottom plate (Falcon; Becton Dickinson, Franklin Lanes, NJ) containing anti-CR1 mAbs (either 1B4 or 7G9) as capture agents. Naive macrophages and macrophages that were reacted with naive E were also lysed as described. The cell lysates were added to appropriate wells and incubated overnight at 4°C. After aspiration of the macrophage lysates, the plate was washed five times with an automated Titertek microplate washer (Titertek Instruments, Huntsville, AL) and was subsequently blocked for 15 min at 37°C with sterile filtered PBS-Tween containing 1% BSA. Then 100 µl of a solution containing 0.1 µg of a different, biotinylated anti-CR1 mAb (b-HB8592) were added to each well, and the plate was incubated for 1 h at 37°C. After a wash, 100 µl of NA-HRP/3000 were added to each well, and the plate was incubated for 30 min at 37°C. After another wash, the plate was developed for 15 min at room temperature using an o-phenylenediamine developing solution, and development was terminated by addition of 50 µl of a 1 M H2SO4 solution. Absorbance readings were taken at 492 nm using a Titertek Multiskan Plus microplate reader.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Loss/gain of IC components

Transfer of the {phi}X174 component of the IC from 51Cr-labeled E to P388D1 cells was investigated by incubating the 51Cr-labeled E-HP-125I-{phi}X174 IC with macrophages at a cellular ratio of 10:1. Analysis by RIA (Fig. 1Go) demonstrated that those E reacted with macrophages lost approximately one-half of the bound 125I counts during the 15-min transfer, whereas E subjected to a mock transfer with media alone lost only 10% of bound 125I counts. In addition, the acceptor macrophages took up a large fraction of the counts released from the E, whereas the media from the mock reaction contained few counts. The absolute number of 125I counts lost from the E during the transfer reaction were somewhat larger than the number taken up by the macrophages, and this difference could be caused by losses due to handling and/or processing and phagocytosis of the IC by the macrophages (and release of digested material) following the transfer. In fact, ~4,000 counts were released into the media after transfer, and 25% of the counts were found to be soluble in 5% trichloroacetic acid. Analysis of 51Cr counts associated with the E at the beginning and at the end of the reaction (after the E were reisolated) confirmed that the E were isolated at high yield and intact after transfer, because 51Cr counts averaged 130,000 ± 2,000 for all samples (transfer, naive E, "mock" transfer, etc.).



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  		FIGURE 1. Transfer of 125I-{phi}X174, bound to 51Cr-labeled human E via HP, to P388D1 macrophages. E were incubated for 15 min with either macrophages (10 E/macrophage, striped bars) or media (dark bars) and then separated. Error bars represent 1 SD in all figures. Substantial loss of 125I is observed for E incubated with macrophages, and most of the lost counts are associated with the macrophages (striped bars, posttransfer). E-associated 51Cr counts remained constant at 130,000 ± 2,000 for all samples (not shown).

 
Because the isolated macrophages took up a large fraction of the released 125I-{phi}X174 in the transfer reaction, we were interested in determining whether this {phi}X174 was bound externally or was internalized by the macrophages. Therefore, additional experiments were performed with both 125I-labeled and unlabeled {phi}X174 in which the P388D1 cells used in the transfer reaction were then probed with 125I-labeled mAb {phi}X51.1 (specific for {phi}X174) which does not compete with mAb 7B7 (used in the HP). The results (Table IGo) again indicate that those E that were reacted with macrophages lost much more 125I-labeled {phi}X174 (35–37%) than did the E in the mock samples (7–12%), and once again macrophages isolated after the transfer reaction contained a substantial amount of {phi}X174 (23–26%), further confirming the findings in the previous experiment. Moreover, the results of the second probing step (Table IIGo) indicate a marked difference in the quantity of 125I counts (specific anti-{phi}X174 mAb) bound to the permeabilized compared with the nonpermeabilized macrophages; {phi}X174 was only located inside those macrophages that had participated in the transfer reaction.


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  		Table I. Transfer of HP-bound 125I-{phi}X174 from E to macrophages: localization of 125I-{phi}X174 before and after IC transfer (25 E/macrophage) within E or macrophage fractions1

 

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  		Table II. Transfer of HP-bound 125I-{phi}X174 from E to macrophages: intracellular localization of {phi}X174 within macrophages after IC transfer1

 
We performed similar experiments to determine whether the HP (labeled with 125I), which was bound to E CR1 and to {phi}X174, was also removed from the 51Cr-labeled E during the transfer reaction and internalized by the P388D1 macrophages. After transfer almost one-half of the HP was removed from the E and approximately three-fourths of the released counts were associated with the macrophages (Table IIIGo). Microscopic examination of the macrophages revealed that there was little, if any, uptake of the E (not shown), and the results obtained by following the 51Cr label (Table IIIGo) further confirm that after the transfer reaction the E are not internalized or associated with the P388D1 cells. This observation is in agreement with previous work in our laboratory (30) which demonstrated that the IgG bound to CR1 does not target E for phagocytosis. To determine whether the transferred HP was located inside or on the outer surface of the macrophages, we performed a similar experiment using unlabeled HP and probed the separated E and intact macrophages (initially incubated at a 10:1 ratio) with FITC-labeled F(ab')2 goat anti-mouse IgG. The results of flow cytometry revealed that ~55% of the HP was removed from the E, but there was no increase in fluorescent signal (above background) of the macrophages after transfer (not shown), thus indicating the HP was not located on the outer surface of the macrophages.


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  		Table III. Transfer of 125I-labeled HP (7G9 x 7B7) from E to P388D1 cells

 
The results presented thus far demonstrate that both the {phi}X174 as well as the HP are removed from the E during the transfer reaction. However, none of the previous experimental protocols addressed whether the E cell surface receptor, CR1, is also lost from the E during the transfer process. Because of the intrinsic low level of CR1 on human E, we used flow cytometry, which has a higher level of sensitivity compared with the RIA, to test for CR1 loss. Specific biotinylated probes (b-7G9, b-Rab@Ms IgG, b-{phi}X51.1) were respectively used to assay for CR1, HP, and {phi}X174 located on E before and following transfer to P388D1 cells (or to media, in controls). The histograms indicate that both {phi}X174 (Fig. 2GoA) and HP (Fig. 2GoB) were removed from the E during the incubation with macrophages, and these later two findings are in agreement with the previous results (see above) obtained by RIA. In addition, Fig. 2GoC demonstrates that CR1 was indeed removed from the E during the transfer process.



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  		FIGURE 2. Flow cytometry experiments demonstrate loss of {phi}X174 (A), HP (B), and CR1 (C) from human E due to the transfer reaction. After incubation with macrophages for 40 min (1 E/5 macrophages), E were separated and respectively probed with appropriate biotinylated Abs specific for {phi}X174, HP, and CR1, followed by FITC-labeled NA. Lines represent samples as follows: naive E (not probed) (black), pre-transfer E-HP-{phi}X174 (red), posttransfer E-HP-{phi}X174 (blue). An E-HP-{phi}X174 sample (green) that was incubated in the presence of media alone was also included as a control. In C, MESF values are as follows: naive E (not probed) (black line), 1190; pretransfer E-HP-{phi}X174 (red line), 3530; posttransfer E-HP-{phi}X174 (blue line), 2450; "mock" transfer E-HP-{phi}X174 (green line), 3550.

 
We used an ELISA capture assay (see Materials and Methods) to determine whether CR1 which was lost from the E surface during the transfer reaction can be demonstrated to be associated with the macrophages. P388D1 cells were isolated following different transfer time points, and the washed cells were lysed and analyzed for the presence of CR1 by ELISA. The results, using wells coated with anti-CR1 capture mAb 1B4, demonstrate that E CR1 was indeed taken up by the macrophages with a maximum occurring after 30 min (Fig. 3Go, filled triangles), and that nearly one-half of the transfer occurred within the first 10 min of the reaction. We note that far less CR1 was found within the macrophages when the capture anti-CR1 mAb was 7G9, which was the same mAb used to prepare the HP (Fig. 3Go, filled circles). When macrophages were reacted with naive human E under otherwise identical conditions, CR1 was not found in the lysed macrophages after the transfer protocol. Finally, flow cytometry experiments with an FITC-labeled anti-CR1 mAb (1B4) failed to demonstrate any CR1 located on the outside of the intact (not permeabilized) macrophages after the transfer reaction (not shown). In the presence of excess mouse IgG (to block Fc receptors), the net fluorescent signal (MESF) after probing with mAb 1B4 was equal to the signal obtained for an irrelevant isotype matched control. In summary, our results demonstrate that CR1, HP, and {phi}X174 are all transferred from E to macrophages, but all three components are located only on the inside of the acceptor cells.



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  		FIGURE 3. E CR1 can be demonstrated to be located within the macrophage fraction after the transfer reaction. E-HP-{phi}X174 complexes (or naive E) and macrophages were incubated (10 E/macrophage) for between 10 and 40 min and were then separated. The macrophages were lysed, and the lysates were assayed for E CR1 in a capture ELISA. Anti-CR1 mAbs used as capture molecules were either 1B4 (filled triangles) or 7G9 (filled circles) as the capture Ab. Levels of E CR1 contained within those macrophages reacted with naive E are represented by the open symbols.

 
We next investigated whether a human monocytic cell line could also be used to study the transfer reaction, and we also examined the potential role of complement in the process. IC consisting of HP, {phi}X174, and an anti-{phi}X174 mAb were bound to human E. After a wash, the E were either opsonized with complement (by incubation with human serum) and washed or simply held in buffer on ice, and then both E samples were incubated with U937 cells. We used flow cytometry to confirm that C3b was indeed bound to the E that were opsonized with complement (not shown). In the absence of complement, a large fraction of the {phi}X174 was removed from the E (Fig. 4Go, black bars) by the U937 cells. The E samples that were opsonized with complement (striped bars) showed less transfer than the samples that were not opsonized with complement (Fig. 4Go, compare E-IC-C3b with E-IC (transfer bars)).



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  		FIGURE 4. Transfer of IC from E CR1 to U937 acceptor cells. E containing bound HP-{phi}X174-anti-{phi}X174 IC (dark bars) or complement-opsonized IC (striped bars) were left on ice (initial) or were incubated for 1 h with media (mock) or with U937 cells (transfer). Following cell separation, E were probed with an FITC-labeled mAb specific for {phi}X174. The MESF value for naive, unprobed E is 260. The MESF value for background binding of FITC-anti-{phi}X174 to naive E is 290.

 
Blocking of IC transfer

We next determined whether certain properties of the IC or macrophages, which when eliminated or blocked, would inhibit transfer of the IC components. To address the importance of Fc receptors in the transfer, we first tested whether HP lacking Fc regions could still facilitate transfer. The results of flow cytometry analyses (Fig. 5Go) indicate that when the {phi}X174 was bound to E via the F(ab')2 HP, there was apparently no recognition by or transfer to the P388D1 cells. However, when additional intact IgG mAb to {phi}X174 (7B7) was added to the E-bound F(ab')2 HP/{phi}X174 complex and was allowed to bind to free sites on the {phi}X174, >85% of the IC were stripped from the E in the presence of macrophages (dark bar on the right side of Fig. 5Go). In the absence of macrophages, there was little, if any, loss of {phi}X174 for the F(ab')2 HP complex in the presence or absence of intact mAb 7B7. These results suggest that macrophage Fc receptors play an important role in transfer. To test this hypothesis further, we used a mAb, 2.4G2, which is known to specifically bind to and block mouse Fc{gamma}RII/III (24). As seen in the RIA in Fig. 6Go, when a high input of mAb 2.4G2 was added to the P388D1 cells (crosshatched bar), transfer was reduced nearly to background levels (black bar). Macrophage samples that were treated with 20-fold less blocking mAb were still partially inhibited from facilitating transfer. These experiments were also performed with 51Cr-labeled E, and once again we found no evidence for trapping or phagocytosis of the E by the macrophages (not shown).



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  		FIGURE 5. Fc portions of Abs are required for IC transfer. FITC-labeled {phi}X174 was bound to monkey E via F(ab')2 HP ± an additional whole IgG anti-{phi}X174 mAb. These E were incubated with macrophages (2 E/macrophage) or media for 30 min, and after separation the E were analyzed by flow cytometry. The MESF value for naive E is 460.

 


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  		FIGURE 6. MAb 2.4G2, specific for mouse Fc{gamma}RII/III, inhibits the transfer reaction. 51Cr-labeled human E were opsonized with HP and 125I-labeled {phi}X174. E and macrophages (±mAb 2.4G2) were incubated for 40 min (2 E/macrophage), and after separation, E were counted simultaneously for 125I and 51Cr.

 
We next investigated how the method of binding {phi}X174 to the E might influence the transfer process, and whether macrophage membrane reorganization might play a role in the reaction. FITC-labeled {phi}X174 was bound to human E by two different protocols (see Materials and Methods). In the first procedure, E were incubated with saturating levels of HP, and after a wash, {phi}X174 was bound to the E-HP (Fig. 7Go; A and C). Alternatively, {phi}X174 was first mixed with the E, and then HP was added to give solution phase binding (Fig. 7Go, B and D). Finally, after the binding reactions, additional anti-{phi}X174 mAb (7A4) was added to the E-IC to further opsonize "free" sites on the E-bound {phi}X174 (Fig. 7Go, C and D). Although less {phi}X174 was bound to E after the solution phase binding protocol (MESF levels average ~1300 in B and D, compared with MESF levels of ~2100 in A and C), the P388D1 cells could remove the {phi}X174 substrate from the E in both cases. Moreover, binding of an additional mAb to E-bound {phi}X174 facilitated a higher level of transfer (compare Fig. 7Go, C and D with Fig. 7Go, A and B). Finally, we found that the transfer reaction was partially inhibited when macrophages were incubated with saturating amounts of cytochalasin D (an inhibitor of actin polymerization (31, 32)) before the addition of E-IC (horizontal striped bars).



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  		FIGURE 7. Cytochalasin D partially inhibits the transfer reaction. FITC-labeled {phi}X174 was bound to E via HP under several experimental conditions (see Materials and Methods), and then an additional anti-{phi}X174 mAb was bound to the E (C and D). The E were incubated for 60 min with media or with macrophages (1 E/macrophage) either in the presence or absence of cytochalasin D (CytoD). Flow cytometry was performed on unseparated gated cell populations.

 
Preliminary investigation into the reason for limited amount of IC transfer

We designed an experiment to learn whether the amount of IC that can be removed from the surface of the E by macrophages is limited and, if so, which cell type is restricting. In this paradigm, both cell types (E and P388D1 cells) were used in two sequential transfer reactions. The first transfer reaction has already been described (Tables I and II, RIA), and we have designated the cells isolated after this reaction as "used." After the first transfer reaction, an additional transfer was performed by the addition of another aliquot of fresh E-HP-{phi}X174 to the used macrophages; similarly, another aliquot of fresh macrophages was added to the used E. At the start of the experiment and after each reaction, the amount of 125I associated with each cell type was determined, and the percentage of transfer is reported. In addition, a mock transfer was also performed to gauge the amount of IC lost from the E in the absence of macrophages.

The results in Table IVGo demonstrate that the percent of {phi}X174 removed from the E-IC in the first reaction (42%) is approximately equal to the percent removed from a fresh set of E-IC by the "used" macrophages during a second reaction (37–44%), and the percent uptake by the macrophages is also similar in both reactions (an average of 29.5% compared with an average of 23.5%). These data imply that the amount of transfer that occurs is not limited by the macrophages, because these cells were able to facilitate approximately the same level of transfer during both reactions.


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  		Table IV. Macrophage saturation is not the reason for the limited amount of 125I-{phi}X174 removed from the E during the transfer reactions

 
In contrast, the E that were involved in two sequential transfer reactions lost 42% of their bound IC during the first reaction but lost only 19% (Table IVGo) during the second transfer reaction with fresh macrophages. In addition, the percent gain of IC by the macrophages that were reacted with used E-IC was less than one-half of the percent taken up by those macrophages that were reacted with fresh E-IC. Finally, we note that in these experiments the E were again labeled with 51Cr and that even under conditions in which E were subjected to two transfer reactions, there was no evidence for sequestration or internalization of the E by the macrophages: 51Cr counts associated with the E remained constant at 125,000 ± 5,000 throughout the procedures.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of the present series of investigations was to develop a reliable in vitro model that could be used to study the mechanism of the reaction by which IC bound to E CR1 via HP are transferred to acceptor macrophages. Our previous work in monkey model systems has indicated that these E-bound IC, generated in the circulation by infusion of {phi}X174 followed by specific HP, are indeed removed from the E and cleared to the liver (15). Studies with other systems in monkey models suggested that loss of CR1 from the E is a key required step in this reaction (33, 34). We chose to focus primarily on a mouse macrophage cell line to eliminate the potential confounding effect of human CR1 located on the acceptor macrophage. We also conducted experiments using E-IC (±C3b) and U937 cells to demonstrate transfer to a human cell line. Also, in other experiments using P388D1 cells, we were able to eliminate any contributions from complement fragments because there was an identical amount of IC transfer (not shown) to macrophages in media containing fresh or heat-inactivated FCS. This system has allowed us to demonstrate that Fc receptors alone can facilitate transfer.

The experimental data (Figs. 1Go and 2Go and Tables I to III), which were obtained in independent flow cytometry and RIA experiments, confirms the basic tenets of the transfer reaction. E-bound HP and {phi}X174 are indeed removed from the E and taken up by the macrophages, and the combination of permeabilization studies and flow cytometric analyses clearly demonstrates that both of these components are internalized by the macrophages. Moreover, we find that not only is CR1 removed from the E during transfer (Fig. 2GoC) but also CR1 can indeed be located within the macrophage fraction (Fig. 3Go). CR1 is presumably still associated with the HP after internalization, because the cognate mAb used to prepare the HP (7G9) is considerably less effective as a capture mAb than mAb 1B4, which binds to CR1 at a different site than 7G9 (Fig. 3Go) (19).

Several lines of evidence suggest that Fc receptors on the macrophages play an important role in transfer of E-bound IC. First, robust transfer is demonstrable in the absence of complement. In addition, use of mAb 2.4G2, specific for Fc{gamma}RII/III of the mouse (and possibly capable of engaging Fc{gamma}RI as an intact IgG molecule (35)) leads to almost complete inhibition of transfer (Fig. 6Go). The fact that IC prepared with HP constructed from F(ab')2 fragments were not transferred unless whole IgG mAb was added to the system (Fig. 5Go) provides further support for an Fc-mediated reaction. Reorganization of the macrophage membrane (presumably mediated by cross-linked Fc receptors) appears to play a role in transfer, because treatment of macrophages with cytochalasin D (known to inhibit actin polymerization) (31, 32) partially inhibits removal of IC from the E (Fig. 7Go).

In a primate system, complement activation and complement receptors on the acceptor cell may also participate in the transfer reaction (2, 3, 5, 6). However, in experiments using U937 cells (human), we found that opsonization of the E-IC with C3b significantly blocked transfer of the {phi}X174 (Fig. 4Go). This blockade may have occurred due to masking of the sites on the Fc portion of IgG (by C3b) which are otherwise recognized by the Fc{gamma} receptors. In several in vivo systems complement activation has been reported to slow the rate of clearance of IgG-containing IC from the circulation (6, 36). In fact, Brown has also suggested that this effect may be mediated by C3b blocking of Fc regions (37). Furthermore, the C3b deposited on the IC may have enhanced binding of the IC to the E by recruiting additional CR1. This effect would also tend to inhibit the transfer reaction, as presumably more CR1 molecules would have to be cleaved to allow release of the IC. In any event, we note that the E-bound HP/{phi}X174 IC are rapidly cleared from the circulation in complement-replete monkey models (15, 16); however, we do not know whether complement affects the rate of clearance. Studies with monkeys treated with cobra venom factor could address this question.

The results of the double transfer experiments (Table IVGo) suggest that in the present model system there may be factors that limit the amount of IC that can be removed from the E. The limiting factors do not appear to be associated with saturation of the processing capacity of the acceptor macrophages, because "used" macrophages that were incubated with fresh E-IC were able to remove as much IC (42%) from the E-IC as did fresh macrophages. Rather, the results of this experiment suggest that in the present experimental system not all of the E-bound IC can be easily removed from the E. The reason for this phenomenon remains to be elucidated but may be explained as follows. Recognition of E-bound IC by Fc receptors appears to play a major role in the transfer reaction, and possibly some of the {phi}X174-IgG HP complexes are bound to E CR1 under conditions in which the local density of IgG is too low to allow for recognition by Fc receptors on the macrophages. That is, the efficiency and rate of the transfer process may depend on the number of Fc regions in the E-bound IC that are engaged by receptors on the macrophage. Thus, only those IC with a sufficiently high local density of IgG in the IC will be easily transferred. The results of the experiment illustrated in Fig. 7Go (C and D) support this interpretation; when additional anti-{phi}X174 mAb was added to the E-IC (to enhance IgG-mediated opsonization and Fc recognition), transfer was enhanced. We note that clearance of model IC from the circulation of animals does depend on the size of the IC and/or the number of IgG per IC. Larger IC are cleared more rapidly, and this finding is generally believed to be related to the efficiency of Fc recognition (7, 38, 39, 40, 41).

The fact that we did not achieve complete transfer of the IC from the E (see above) also suggests that the Fc-mediated process is not 100% efficient in mediating transfer. However, based on our findings we suggest that the transfer reaction can occur as follows. The E-bound IC is recognized and bound by Fc receptors on the acceptor macrophage. Close juxtaposition of the two cells (perhaps partially mediated by Fc receptor movement) leads to cleavage of CR1 by a macrophage protease and results in detachment of the IC from the E. The released IC (containing CR1) would then be phagocytosed by the macrophage (a well-described reaction that requires membrane reorganization), and the E which had "lost" the IC would be spared during the reaction. Direct demonstration after transfer of a residual membrane-associated fragment of CR1 still associated with the E will be required to confirm the hypothesis. There is certainly precedence for such a proteolytic reaction. In fact, a number of membrane-associated proteases located on macrophages and other cells have been described (42, 43). Furthermore, many of these proteases are known to be able to cleave certain membrane-associated proteins, which like CR1 are composed of the short consensus repeat structural motif (44, 45, 46, 47). Finally, studies in monkey models and in humans of the processing of substrates bound to E CR1 in the circulation have suggested that loss and/or proteolysis of CR1 is a key step in the clearance reaction (5, 6, 33, 34). The present series of in vitro investigations provide strong support for this model.


   Acknowledgments
 
We thank Professor Nino Incardona for the gift of the bacteriophage {phi}X174.


   Footnotes
 
1 This work was supported by the Defense Advanced Research Projects Agency, Order MDA972–96-K-0001 under Contract MDA972-96-K-003, and by National Institutes of Health Grant AR43307. Back

2 Address correspondence and reprint requests to Dr. Ronald P. Taylor, Box 440, Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, VA 22908. Back

3 Abbreviations used in this paper: IC, immune complex; CR1, complement receptor 1; HP, heteropolymer; {phi}X174, bacteriophage {phi}X174; P388D1, mouse macrophage tumor cell line; 2.4G2, mouse mAb specific for Fc{gamma}RII/III; NA, neutralite avidin; TX100, Triton X-100; MESF, mean equivalent soluble fluorochromes; Fc{gamma}RII/III, Fc receptors. Back

Received for publication June 14, 1999. Accepted for publication December 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nelson, R. A.. 1953. The immune adherence phenomenon: an immunologically specific reaction between micro-organisms and erythrocytes leading to enhanced phagocytosis. Science 118:733.[Free Full Text]
  2. Schifferli, J. A., R. P. Taylor. 1989. Physiological and pathological aspects of circulating immune complexes. Kidney Int. 35:993.[Medline]
  3. Cornacoff, J. B., L. A. Hebert, W. L. Smead, M. E. VanAman, D. J. Birmingham, F. J. Waxman. 1983. Primate erythrocyte-immune complex-clearing mechanism. J. Clin. Invest. 71:236.
  4. Fearon, D. T.. 1980. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152:20.[Abstract/Free Full Text]
  5. Davies, K. A., V. Hird, S. Stewart, G. B. Sivolapenko, P. Jose, A. A. Epenetos, M. J. Walport. 1990. A study of in vivo immune complex formation and clearance in man. J. Immunol. 144:4613.[Abstract]
  6. Davies, K. A., A. M. Peters, H. L. Beynon, M. J. Walport. 1992. Immune complex processing in patients with systemic lupus erythematosus. In vivo imaging and clearance studies. J. Clin. Invest. 90:2075.
  7. Bogers, W. M., R. K. Stad, L. A. Van Es, M. R. Daha. 1992. Both Kupffer cells and liver endothelial cells play an important role in the clearance of IgA and IgG immune complexes. Res. Immunol 143:219.[Medline]
  8. Nishi, T., A. K. Bhan, A. B. Collins, R. T. McCluskey. 1981. Effect of circulating immune complexes on Fc and C3 receptors of Kupffer cells in vivo. Lab. Invest. 44:442.[Medline]
  9. Edberg, J. C., G. A. Kujala, R. P. Taylor. 1987. Rapid immune adherence reactivity of nascent, soluble antibody/DNA immune complexes in the circulation. J. Immunol. 139:1240.[Abstract]
  10. Cosio, F. G., X. P. Shen, D. J. Birmingham, M. Van Aman, L. A. Hebert. 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.[Abstract]
  11. Madi, N., J. P. Paccaud, G. Steiger, J. A. Schifferli. 1991. Immune complex binding efficiency of erythrocyte complement receptor 1 (CR1). Clin. Exp. Immunol. 84:9.[Medline]
  12. Taylor, R. P., W. M. Sutherland, C. J. Reist, D. J. Webb, E. L. Wright, R. H. Labuguen. 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.[Abstract/Free Full Text]
  13. Kuhn, S. E., A. Nardin, P. E. Klebba, R. P. Taylor. 1998. Escherichia coli bound to the primate erythrocyte complement receptor via bispecific monoclonal antibodies are transferred to and phagocytosed by human monocytes in an in vitro model. J. Immunol. 160:5088.[Abstract/Free Full Text]
  14. Nardin, A., W. M. Sutherland, M. Hevey, A. Schmaljohn, R. P. Taylor. 1998. Quantitative studies of heteropolymer-mediated binding of inactivated Marburg virus to the complement receptor on primate erythrocytes. J. Immunol. Methods 211:21.[Medline]
  15. Taylor, R. P., E. N. Martin, M. L. Reinagel, A. Nardin, M. Craig, Q. Choice, R. Schlimgen, S. Greenbaum, N. L. Incardona, H. D. Ochs. 1997. Bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model. J. Immunol. 159:4035.[Abstract]
  16. Taylor, R. P., W. M. Sutherland, E. N. Martin, P. J. Ferguson, M. L. Reinagel, E. Gilbert, K. Lopez, N. L. Incardona, H. D. Ochs. 1997. Bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model. J. Immunol. 158:842.[Abstract]
  17. Emlen, W., V. Carl, G. Burdick. 1992. Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes. Clin. Exp. Immunol. 89:8.[Medline]
  18. Sundstrom, C., K. Nilsson. 1976. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17:565.[Medline]
  19. Ferguson, P. J., C. J. Reist, E. N. Martin, C. Johnson, K. L. Greene, S. Kuhn, J. Niebur, W. Emlen, R. P. Taylor. 1995. Antigen-based heteropolymers: a potential therapy for binding and clearing autoantibodies via erythrocyte CR1. Arthritis Rheum. 38:190.[Medline]
  20. Tausk, F. A., A. McCutchan, P. Spechko, R. D. Schreiber, I. Gigli. 1986. Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men in varying risk groups for acquired immune deficiency syndrome. J. Clin. Invest. 78:977.
  21. O’Shea, J. J., E. J. Brown, B. E. Seligmann, J. A. Metcalf, M. M. Frank, J. I. Gallin. 1985. Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils. J. Immunol. 134:2580.[Abstract]
  22. Edberg, J. C., E. Wright, R. P. Taylor. 1987. Quantitative analyses of the binding of soluble complement-fixing antibody/dsDNA immune complexes to CR1 on human red blood cells. J. Immunol. 139:3739.[Abstract]
  23. Segal, D. M., B. Bast. 1995. Production of bispecific antibodies. ed. Current Protocols in Immunology 2.13.1. Wiley, New York.
  24. Unkeless, J. C.. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150:580.[Abstract/Free Full Text]
  25. Fraker, P. J., Jr J. C. Speck. 1978. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80:849.[Medline]
  26. Bayer, E. A., M. Wilchek. 1990. Protein biotinylation. Methods Enzymol. 184:138.[Medline]
  27. Harlow, E., D. Lane. 1988. Antibodies: a Laboratory Manual 354. Cold Spring Harbor Laboratory, Cold Spring Harbor.
  28. Reist, C. J., M. J. Combs, B. Y. Croft, R. P. Taylor. 1993. Antigens pre-bound to the primate erythrocyte complement receptor via cross-linked bispecific monoclonal antibody heteropolymers are rapidly cleared from the circulation. Eur. J. Immunol. 23:3021.[Medline]
  29. Edberg, J. C., L. Tosic, E. L. Wright, W. M. Sutherland, R. P. Taylor. 1988. Quantitative analyses of the relationship between C3 consumption, C3b capture, and immune adherence of complement-fixing antibody/DNA immune complexes. J. Immunol. 141:4258.[Abstract]
  30. Reinagel, M. L., M. Gezen, P. J. Ferguson, S. Kuhn, E. N. Martin, R. P. Taylor. 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.[Abstract/Free Full Text]
  31. Brenner, S. L., E. D. Korn. 1979. Substoichiometric concentrations of cytochalasin D inhibit actin polymerization: additional evidence for an F-actin treadmill. J. Biol. Chem. 254:9982.[Free Full Text]
  32. Flanagan, M. D., S. Lin. 1980. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J. Biol. Chem. 255:835.[Abstract/Free Full Text]
  33. Reist, C. J., H. Y. Liang, D. Denny, E. N. Martin, W. M. Scheld, R. P. Taylor. 1994. Cross-linked bispecific monoclonal antibody heteropolymers facilitate the clearance of human IgM from the circulation of squirrel monkeys. Eur. J. Immunol. 24:2018.[Medline]
  34. Taylor, R. P., P. J. Ferguson, E. N. Martin, J. Cooke, K. L. Greene, K. Grinspun, M. Guttman, S. Kuhn. 1997. Immune complexes bound to the 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. Immunopathol. 82:49.[Medline]
  35. Kurlander, R. J., D. M. Ellison, J. Hall. 1984. The blockade of Fc receptor-mediated clearance of immune complexes in vivo by a monoclonal antibody (2. 4G2) directed against Fc receptors on murine leukocytes. J. Immunol. 133:855.[Abstract]
  36. Waxman, F. J., L. A. Hebert, J. B. Cornacoff, M. E. VanAman, W. L. Smead, E. H. Kraut, D. J. Birmingham, J. M. Taguiam. 1984. Complement depletion accelerates the clearance of immune complexes from the circulation of primates. J. Clin. Invest. 74:1329.
  37. Brown, E. J.. 1998. Cooperation between IgG Fc receptors and complement receptors in host defence. ed. The Immunoglobulin Receptors and Their Physiological and Pathological Roles in Immunity 141. Kluwer, Boston.
  38. Weigle, W. O.. 1958. Elimination of antigen-antibody complexes from the sera of rabbits. J. Immunol. 81:204.
  39. Mannik, M., M. P. Arend, A. P. Hall, B. C. Gilliland. 1971. Studies on antigen-antibody complexes. I. Elimination of soluble complexes from rabbit circulation. J. Exp. Med. 133:713.[Abstract]
  40. Haakenstad, A. O., M. Mannik. 1976. The disappearance kinetics of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice. Lab. Invest. 35:283.[Medline]
  41. Finbloom, D. S., P. H. Plotz. 1979. Studies of reticuloendothelial function in the mouse with model immune complexes. I. Serum clearance and tissue uptake in normal C3H mice. J. Immunol. 123:1594.[Abstract/Free Full Text]
  42. Bazil, V., J. L. Strominger. 1994. Metalloprotease and serine protease are involved in cleavage of CD43, CD44, and CD16 from stimulated human granulocytes: induction of cleavage of L-selectin via CD16. J. Immunol. 152:1314.[Abstract]
  43. Bauvois, B., J. Sanceau, J. Wietzerbin. 1992. Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-{alpha}. Eur. J. Immunol. 22:923.[Medline]
  44. Kahn, J., R. H. Ingraham, F. Shirley, G. I. Migaki, T. K. Kishimoto. 1994. Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin. J. Cell Biol. 125:461.[Abstract/Free Full Text]
  45. Chen, A., P. Engel, T. F. Tedder. 1995. Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. J. Exp. Med. 182:519.[Abstract/Free Full Text]
  46. Preece, G., G. Murphy, A. Ager. 1996. Metalloproteinase-mediated regulation of L-selectin levels on leukocytes. J. Biol. Chem. 271:11634.[Abstract/Free Full Text]
  47. Fremeaux-Bacchi, V., I. Bernard, F. Maillet, J. C. Mani, M. Fontaine, J. Y. Bonnefoy, M. D. Kazatchkine, E. Fischer. 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.[Medline]



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