The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuhn, S. E.
Right arrow Articles by Taylor, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuhn, S. E.
Right arrow Articles by Taylor, R. P.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CYTOCHALASIN D
*ETHYLENEDIAMINE TETRAACETIC ACID
The Journal of Immunology, 1998, 160: 5088-5097.
Copyright © 1998 by The American Association of Immunologists

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 Model1

Susan E. Kuhn*, Alessandra Nardin*, Philip E. Klebba{dagger} and Ronald P. Taylor2,*

* Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, VA 22908; and {dagger} Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have prepared cross-linked, bispecific mAb complexes (heteropolymers) that facilitate rapid and quantitative binding of a prototype pathogen, Escherichia coli, to the complement receptor (CR1) on primate erythrocytes. Incubation of the erythrocyte-heteropolymer-E. coli complexes with freshly isolated human mononuclear cells leads to rapid removal of the E. coli from the erythrocytes, and phagocytosis and killing of the bacteria. The erythrocytes are not lysed or phagocytosed during this transfer reaction, but both heteropolymer and CR1 are removed from the erythrocytes along with the E. coli. These findings parallel observations made in previous in vivo experiments in which heteropolymers were used to facilitate clearance of innocuous prototype pathogens in a monkey model. It should now be possible to extend the heteropolymer paradigm to a live pathogen in a primate model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In 1953, Nelson proposed an immunologic role for primate erythrocytes (E)3 based on his investigations of the immune adherence reaction (1). He observed that bacteria opsonized with both Abs and complement adhere to E and that this binding leads to enhanced phagocytosis and killing of the micro-organisms. It is now recognized that E binding in this reaction is mediated by complement receptor 1 (CR1), a type I membrane-associated glycoprotein receptor found on primate E and other cells that is specific for C3b/C4b (covalently associated with the opsonized substrates) (2, 3). This enhanced killing of the E-bound and complement-opsonized micro-organisms led Nelson to suggest that E presentation of such bound particulate pathogens could play a role in controlling bacterial infections in the bloodstream. Inherent in this idea is the presumption that once bound to E, particulate pathogens would be more easily taken up and destroyed by appropriate phagocytic cells, which could include neutrophils, monocytes, or fixed tissue macrophages. Studies of the E-immune complex (IC) clearance reaction, initiated 30 yr after Nelson, have extended this reaction to soluble substrates. This work has revealed that a fraction of the soluble Ab-protein Ag IC (nonparticulate) that forms in the circulation can fix complement, bind to E, and then be cleared from the circulation and destroyed in the liver and spleen (3, 4, 5).

We have developed an alternative procedure to bind target pathogens (both micro-organisms and protein Ags) to primate E via CR1 with a very high level of efficiency in the complete absence of complement (6, 7, 8, 9). The method is based on using bispecific mAb complexes that are constructed by cross-linking a mAb specific for CR1 (which serves as a surrogate for C3b) with a mAb specific for the target pathogen. Based on Nelson’s original work and the more widely studied E-based IC clearance phenomenon, we have proposed that these bispecific complexes (heteropolymers (HP); anti-CR1 mAb x anti-pathogen mAb) have the potential to bind both soluble and particulate pathogens to E in the bloodstream and then to present the pathogens to acceptor cells for phagocytosis and destruction. In fact, our in vivo experiments in monkey models have verified that once bound to E CR1 via specific HP, both soluble proteins and a prototype virus are cleared from the circulation and destroyed in the liver by a mechanism quite similar, in many respects, to the E-IC clearance reaction (8, 10, 11).

We have now extended our examination of the HP paradigm to an in vitro model, similar to that examined by Nelson, which in this case focuses on E. coli as a model particulate pathogen. We have used specific HP to bind E. coli to primate E, and we have studied the transfer of this E-bound substrate to human monocytes. The results of these studies, performed in the absence of complement, indicate that Escherichia coli bound to E CR1 via HP are indeed phagocytosed and destroyed by human monocytes. We also demonstrate that this transfer reaction, which includes the concomitant loss of E CR1, shows a striking similarity to the in vivo reaction by which substrates bound to E CR1 are cleared from the circulation in primates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse mAbs and HP

mAb 7G9 (IgG2a) and mAb 44 (IgG2b), respectively specific for CR1 and E. coli, have been reported (8, 10, 12, 13, 14). Other mAbs used and their specificities are as follows: HB8592 (IgG1, anti-CR1) (15), 7B7 (IgG2a, irrelevant specificity) (11), IV.3 (IgG2b, anti-FcRII, Medarex, Annandale, NJ). HP were formed by covalently cross-linking mAb 7G9 or mAb HB8592 with mAb 44 via a thioether bond as previously described (11, 16) and purified by fast protein liquid chromatography. The subfractions corresponding to dimers through tetramers were used in all experiments.

E. coli

The BN1071 strain of E. coli, previously shown to bind mAb 44 due to a high level of expression of the iron-binding protein Fep A (12, 13), was used in all experiments. In certain studies the bacteria were covalently labeled with either FITC (17) or biotin (18). The bacteria were washed and reconstituted in PBS to 1 x 109 cells/ml, and then incubated for 2 h at room temperature with either FITC (Isomer I, Sigma, St. Louis, MO; final concentration of 0.01 mg/ml) or biotin N-hydroxysuccinimide (long arm, Vector Laboratories, Burlingame, CA; final concentration, 5–30 µg/ml). After three washes, the labeled bacteria were stored at 4°C in PBS containing 1% glucose.

Cells

E were obtained from rhesus and stump-tailed macaque monkeys or from normal human donors. Except where explicitly noted (e.g., Fig. 4Go and Table IVGo), monkey E were used in all the experiments. The blood was stored as a 25% dispersion in Alsever’s solution and washed three times in PBS with 1% BSA (BSA/PBS) before use. Mononuclear cells (designated white blood cells (WBC)) were isolated from fresh human blood drawn into EDTA using Ficoll-Hypaque Plus (Pharmacia Biotech, Uppsala, Sweden) (19). They were washed twice in HBSS, diluted to 1 x 108 cells/ml in RPMI 1640, stored on ice, and used within 4 h. The WBC preparations generally contained 15 to 20% monocytes as determined by light scattering (FACS) or probing with FITC-labeled mAb to CD14 (Becton Dickinson, San Jose, CA) and were >90% viable as defined by trypan blue exclusion. WBC from numerous different donors were used during the course of this work, but individual experiments (on a given day) were performed with the WBC of a single donor. Although the quantitative potential of monocytes to facilitate transfer and killing varied from donor to donor, trends were highly consistent across all donors. In transfer studies from human E to human monocytes, both cell types were freshly isolated and used from the same donor.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4. Transfer of E. coli from human E to autologous human monocytes. The experimental design was similar to that described in Figures 2Go and 3Go, except the cells were incubated together for 20 min. A and B represent two independent experiments; in B the uptake of HP-opsonized E. coli by human monocytes in the absence of human E (20-min incubation) is also displayed (dotted line). MESF values for E in A and B are, respectively, 191 and 203 for naive cells, 4,601 and 6,576 for control cells, and 1,193 and 2,661 for the posttransfer sample. MESF values for the monocytes in the transfer reaction in A and B are 3,015 and 2,604 in naive cells and 12,830 and 9,737 for the posttransfer sample. The MESF value for monocytes incubated with HP-opsonized E. coli alone is 36,239.

 

View this table:
[in this window]
[in a new window]
  		Table IV. Monocyte killing of E. coli1

 
E. coli binding to E via HP

HP-mediated binding of E. coli to E was first tested using CFU assays. Between 2.5 x 105 and 2.5 x 107 E. coli were added to a 50% dispersion of 5 x 108 E in BSA/PBS (thus, E were in excess), and then an aliquot of HP (mAb 7G9 x mAb 44) was added. Following a 1- to 30-min incubation at 37°C, sterile PBS was added, and the samples were centrifuged at 500 rpm for 10 min, a speed and duration selected to pellet the E but minimize pelleting of unbound E. coli. CFU were then measured on multiple dilutions of lysed pellets and supernatants. Specificity of binding was verified by either omitting the HP or by performing the experiments with HP in the presence of an excess of the constituent monomeric components of the HP (excess mAb 7G9 or mAb 44). Control experiments were performed in the absence of E to test for possible precipitation of the bacteria due to cross-linking. In preparation for the transfer experiments, HP-mediated binding of FITC-labeled E. coli to E was tested under conditions where the bacteria were in excess. FITC-labeled E. coli were added to a 1% dispersion of E in BSA/PBS at a 2.5:1 ratio of bacteria to E. Following addition of HP, samples were incubated for 15 min at 37°C, and after a centrifugation step, binding (typically 30–40%) was determined by measuring the fluorescence intensity (unbound bacteria) of the supernatants at 520 nm (excitation length 490 nm) in a fluorometer.

Transfer of E-bound E. coli to monocytes

FITC-labeled E. coli were bound to either monkey or human E via HP, and the E were then washed twice to remove any residual unbound bacteria. Control samples for transfer (E plus FITC-labeled E. coli alone) were prepared without HP by adding to E the amount of FITC-labeled E. coli that was found to bind to E in the presence of HP. These samples were not washed. All E samples were resuspended to 1% before use.

E were mixed with WBC to achieve an E:monocyte ratio (presuming 20% of the WBC were monocytes) between 1:1 and 10:1 and were reconstituted to a final volume of approximately 250 µl with RPMI. The samples were then incubated for 2 to 20 min at 37°C with end-over-end shaking. Following the incubation, the samples were immediately placed on ice and suspended in cold RPMI. The E and WBC were separated using a Percoll density gradient (8, 20) and washed in PBS, and the isolated cells were separately suspended in 1% paraformaldehyde for FACS analysis. To test for nonspecific loss of E. coli from E due to the processing steps, E-HP-E. coli complexes were simply mixed with RPMI (no WBC) but otherwise processed in the same fashion.

The fate of the HP and monkey E CR1 were also examined during the course of the transfer reaction. Biotinylated E. coli were substituted for FITC-labeled E. coli, and following transfer and cell separation, the E were independently probed with FITC-labeled anti-CR1 mAb HB8592, FITC-labeled goat anti-mouse IgG (to probe for HP), or FITC-avidin (to probe for biotinylated E. coli). mAb HB8592 was selected because binding of this mAb to monkey E is the same for both naive and HP (mAb 7G9 x mAb 44)-opsonized E (21). A similar approach was used to analyze human E for loss of CR1 after the transfer reaction. However, due to inhibition of binding of mAb HB8592 to human E-HP-E. coli complexes if the mAb 7G9 x mAb 44 HP was used (not observed with the monkey E), in this case the HP were prepared with anti-CR1 mAb HB8592 and mAb 44, and CR1 was measured by probing with FITC-labeled anti-CR1 mAb 7G9. No inhibition of binding of mAb 7G9 was evident for this combination of reagents.

FACS analysis was conducted on a Becton Dickinson FACScan using CellQuest software. Monocytes were selected from the WBC population using light scattering. During the course of each experiment, FITC standards (Quantum 26 Beads, Flow Cytometry Standards, San Juan, Puerto Rico) were run with the samples, allowing the mean fluorescence data to be normalized to molecules of equivalent soluble fluorochrome (MESF).

Internalization of E. coli by WBC

Transfer was conducted with E-HP-biotinylated-E. coli complexes incubated with WBC. After the separation of E and WBC, the WBC samples were divided into two aliquots; one aliquot was held on ice, while the other was permeabilized using Becton Dickinson FACS Permeabilizing Solution. Both aliquots were then probed simultaneously with Texas Red-avidin (Biomeda, Foster City, CA) and FITC-anti-CD14 (Becton Dickinson), washed multiple times in PBS, and fixed with paraformaldehyde. Slides were prepared for fluorescent microscopy using an antiquenching agent (FluoroGuard, Bio-Rad, Hercules, CA), and the cells were counted and photographed on an Olympus BX60 microscope at x1000 magnification. The percent positive cells is defined as the number of Texas Red-positive cells (with bound biotinylated E. coli) per 100 FITC-positive (CD14-positive) cells. The phagocytic index refers to the number of biotinylated E. coli bound per 100 CD14-positive cells. Typically, 200 CD14-positive cells were counted per experimental determination.

Inhibition of transfer

The WBC were treated with a variety of reagents in an attempt to block transfer of E. coli from E to monocytes. For example, WBC were preincubated with 40 µg/ml mAb IV.3 (anti-FcRII), 1 mg/ml mAb 7G9 (anti-CR1), or 1 mg/ml mAb 7B7 (isotype-matched control) for 4 h on ice to block monocyte receptors. Protease activity was inhibited by incubating WBC in buffer containing either a protease inhibitor mixture (Sigma General Use Protease Inhibitor Mixture; broad specificity against serine, cysteine, aspartic, and metalloproteases, augmented with pepstatin A) or 10 mM EDTA for 30 min on ice. The importance of Ca2+ was tested by including 10 mM Ca2+ in one of the EDTA samples (22). Following these incubations, opsonized E were added directly to the incubation mixture, and a transfer experiment was performed as described above.

The potential role of various monocyte cellular processes in transfer was examined by incubating the WBC in 5 µg/ml cytochalasin D (23), 10 µM colchecine (24), 10 mM EDTA, 200 µM genistein (25), 50 µM sphingosine (23), or 0.1 µM staurosporine (23) (all purchased from Sigma) for 30 min at 37°C. Following these incubations, opsonized E were added directly to the incubation mixture, and an internalization experiment was performed as described above. The concentration of inhibitor was maintained throughout all washes.

Monocyte-mediated killing of E. coli bound to E

E. coli were bound to E via HP (the E were washed twice after binding) or were simply mixed with E in solution (and not washed) in E. coli:E ratios varying between 1:1 and 1:100. These samples were then added to WBC, with final E. coli:monocyte ratios ranging from 1:1 to 1:50, and incubated for 1 h at 37°C. Following the incubation, the samples were centrifuged for 15 min at 4500 rpm to pellet all E. coli and cells. The supernatants were removed, and the pelleted cells were lysed with distilled water. CFU assays were performed on multiple dilutions of the lysates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E binding of E. coli via HP

HP specific for E CR1 and E. coli were prepared and tested for their ability to facilitate binding of E. coli to E. After a slow centrifugation step to pellet the E, binding was examined by measuring CFU in the pellets and supernatants. The results in Table IGo demonstrate that the HP (anti-CR1 mAb 7G9 x anti-E. coli mAb 44) was able to promote rapid and efficient binding of bacteria to E; typically 2 log units or more of E. coli were removed from solution and bound to the surface of the E in <15 min, with substantial binding in as little as 2 min. Virtually all specific binding was eliminated in the absence of HP or in the presence of excess mAb 7G9 or mAb 44, proving that binding was HP mediated and specific for both CR1 and E. coli (Fig. 1Go). In the absence of E, there was a small amount of E. coli precipitation, possibly due to complexes formed by cross-linking. FACS analysis confirmed that the HP do mediate binding of FITC-labeled E. coli to both monkey and human E (Figs. 2Go and 4).


View this table:
[in this window]
[in a new window]
  		Table I. HP mediated binding of E. coli to E1

 


View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. Specificity of HP-mediated binding of E. coli to monkey E. E were incubated with E. coli in the absence of HP or in the presence of both HP and an excess of one of its component parts (mAb 7G9 or mAb 44). E were omitted to test for precipitation of the bacteria. Following a 15-min incubation at 37°C, the E were pelleted through a slow centrifugation step, CFU in the pellets and supernatants were measured, and the percentage of E. coli bound was calculated.

 


View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 2. Transfer of E-bound E. coli to human monocytes. FITC-labeled E. coli were bound to E via HP and then the opsonized E were incubated for 5 min at 37°C with WBC. Following the incubation, the E and WBC were separated by a Percoll density gradient and analyzed by FACS. Control samples (bold) were prepared as follows. E-HP-E. coli complexes or WBC were respectively incubated with buffer alone, but otherwise underwent all processing steps. The percentage of cells that are FACS positive (mean fluorescence >15 units) is displayed. A and B represent two independent experiments. Calculations for E of Expt. A: MESF values are 321 for naive E, 1,950 for the control sample (mock transfer), and 730 for the posttransfer sample. The percent loss in E. coli-associated MESF = (1,950 - 730)/(1,950 - 321) = 75%. The original (unprocessed) E-HP-E. coli sample was also 37% FACS positive and had a MESF of 1830. The respective values for E in Expt. B are 426 (naive), 41,889 (control), and 5,753 (posttransfer), thus giving a net loss in E. coli-associated MESF of 87%. MESF values for the monocytes in parts A and B are, respectively, 4,300 and 4,573 for naive cells and 7,537 and 22,350 for the posttransfer sample.

 
Transfer of E-bound E. coli to monocytes

E with HP-bound FITC-labeled E. coli were incubated at 37°C with freshly isolated WBC at an E. coli:E:monocyte ratio (presuming that 20% of the WBC are monocytes) of 0.75:1:1. Following incubation and separation of cells, FACS analysis demonstrated that the E had been partially depleted of bound E. coli, while a subset of the monocytes had taken up the fluorescent bacteria (Fig. 2Go). The rate of the reaction was quite rapid at 37°C, and the majority of transfer occurred within 5 min (Fig. 3Go and Table IIGo). When the transfer reaction was conducted on ice instead of at 37°C, loss of E. coli from E was only partially inhibited, with loss of bacteria from the E after 5 min on ice approximately half the loss observed at 37°C (MESF values: 6049 down to 3722 compared with 7341 down to 2575; Table IIGo). However, uptake of E. coli by monocytes on ice was blocked considerably.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. Kinetics of transfer of E-bound E. coli to human monocytes. E-HP-FITC-E. coli complexes were incubated with WBC for 2, 5, or 20 min on ice or at 37°C. Cells were separated, and E and monocytes were analyzed by FACS. Control samples are shown in bold. See Table IIGo for quantitative analyses of each histogram. Due to a low recovery of cells, the histogram for the E control for 20 min on ice is taken from the comparable 5-min sample.

 

View this table:
[in this window]
[in a new window]
  		Table II. Effects of incubation time and temperature on the transfer reaction1

 
Control experiments were performed in which free FITC-labeled E. coli, E, and WBC were mixed in the above ratio (no HP added), and negligible binding of E. coli to monocytes was observed (results not shown). To ensure that monocyte uptake of FITC-labeled E. coli was specifically facilitated by HP-mediated E binding, further control experiments were performed by substituting either mAb 44 or an irrelevant HP (mAb 7G9 x mAb 7B7) for the E. coli-specific HP. Monocyte uptake remained at background levels after an incubation of 5 min at 37°C (results not shown).

We also conducted a number of experiments in which E. coli were first bound to human E via HP (mAb 7G9 x mAb 44, as with monkey E), and then transfer to autologous monocytes was studied. The results (Fig. 4Go) demonstrate robust transfer in this system as well. We also determined that direct opsonization of E. coli with HP in the absence of E led to a very high level of binding of the bacteria to monocytes (Fig. 4Go) after a 20-min incubation at 37°C. This high level of direct binding is quite reasonable. The input ratio of HP-opsonized E. coli to WBC for this particular system was approximately 3 times higher than the input ratio when E-HP-E. coli complexes were incubated with WBC because only 30 to 40% of the E. coli bound to E (see Materials and Methods).

Spontaneous release of E. coli bound to E via HP was quite low. Monkey and human E samples containing HP-bound E. coli were processed in the control experiments in an identical protocol, except WBC were omitted from the incubation mixtures and retained >90% of bound E. coli, as defined by both FACS-positive cells and mean fluorescence intensities.

A number of in vivo and in vitro studies have demonstrated that E opsonized with IgG Abs bound exclusively to CR1 are not lysed or phagocytosed, even when IC bound to the E are transferred to phagocytes (3, 4, 19, 21, 26). To ensure that there was no E loss in the present system, following incubation of the E-HP-E. coli complexes with WBC, samples were washed and then subjected to conditions that lysed the E while leaving the monocytes intact (19). After a centrifugation step, the OD540 of the resulting supernatants were measured to determine the original E concentrations. Samples incubated with WBC yielded absorbances equal to those incubated in medium alone (WBC omitted), confirming that no loss of E occurred during the HP-mediated transfer (results not shown).

When CR1 and HP levels on the E were measured before and after transfer, FACS analyses indicated that, along with the transfer of the majority of HP-bound E. coli from the E, the majority of both HP and E CR1 were also removed (Fig. 5Go). E with HP alone also sustained losses of the majority of their HP and CR1, although the decreases were not as great as those seen for E with HP-E. coli complexes. These results are consistent with other studies that suggest that a key step in the transfer reaction includes proteolysis of E CR1 by a protease presumably associated with the acceptor cell, a step that would lead to release of the HP-E. coli-CR1 complex to the phagocyte (8, 21, 26, 27). Interestingly, even naive E lost approximately one-third of their CR1 upon incubation with WBC, indicating that for the monkey E/human monocyte system, the putative protease-like activity of the monocytes may be constitutive, although this activity is enhanced in the presence of immune complexes bound to E CR1. Birmingham et al. reported that E CR1 of baboons and cynomolgus monkeys is membrane bound in a glycophosphatidylinositol (GPI) linkage (28). We found that E CR1 of both the rhesus and stump-tailed macaques (but not human) used in the present study has the same anchor (results not shown). Therefore, we attempted to block the transfer reaction in the monkey E system with phospholipase C inhibitors such as neomycin sulfate or p-chloromercuriphenylsulfonic acid. Neomycin sulfate (10 mM) had no effect on the transfer reaction or on the CR1 loss, and p-chloromercuriphenylsulfonic acid caused E lysis (results not shown).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 5. Loss of E. coli, HP, and CR1 from E during transfer. E-HP-biotinylated E. coli were incubated with WBC for 15 min at 37°C. The E were then separated from the WBC and divided into aliquots that were probed with FITC-avidin (to stain the biotinylated E. coli), FITC-anti-mouse IgG (to stain for HP), or FITC-HB8592 (to probe for CR1). Final samples were compared with control samples that were incubated with buffer instead of WBC, but otherwise underwent all processing steps to determine the percent loss in probe-associated MESF. Values represent the mean ± SD for two independent experiments.

 
We also examined human E-HP-E. coli complexes (mixed with autologous WBC) for loss of E. coli, HP, and CR1 after the transfer reaction. HP were prepared with anti-CR1 mAb HB8592, and CR1 was assayed with FITC-labeled anti-CR1 mAb 7G9 (see Materials and Methods). The results of two independent studies (two different blood donors; in all cases results are compared with the mock transfer; no WBC condition) are as follows: loss of E. coli, 68 ± 4%; loss of HP, 48 ± 2%; and loss of CR1, 13 ± 1%. On the average, less than one E. coli was bound per E (see Materials and Methods), and thus a large fraction of total E CR1 could not have been directly involved in binding the bacteria. In addition, only 68% of the bacteria were transferred, and therefore, the relatively small decrease in E CR1 is quite reasonable. Finally, control experiments indicated that there was no constitutive loss of E CR1 when naive human E were incubated with autologous WBC.

Internalization and killing of E. coli

For HP-mediated clearance to be useful therapeutically, HP-pathogen complexes must be removed from E by phagocytes and then internalized and degraded. To assay for internalization, HP-mediated transfer was conducted with biotinylated E. coli, and both permeabilized and nonpermeabilized aliquots of the WBC were probed with Texas Red-avidin. As demonstrated in the fluorescence micrographs in Figure 6Go, monocytes with intact membranes have very few bound E. coli. However, in comparable permeabilized samples it is evident that the majority of the monocytes have internalized substantial amounts of biotinylated E. coli, with some monocytes containing as many as 10 bacteria. Virtually all monocyte-associated bacteria were found to be internalized after both 5 and 15 min of transfer (Table IIIGo).



View larger version (98K):
[in this window]
[in a new window]
  		FIGURE 6. Internalization of E. coli by human monocytes. Biotinylated E. coli were bound to E via HP, and the E were then incubated with WBC for 15 min at 37°C. The WBC were separated from the E and split into two aliquots; one of the aliquots was permeabilized, while the other was held on ice. Both aliquots were then double stained with Texas Red-avidin (to visualize the biotinylated E. coli) and FITC-anti-CD14 (to visualize the monocytes). In permeabilized samples (A andB), the majority of the FITC-stained monocytes display rod-shaped E. coli. Some of the E. coli lie out of the focal plane and therefore have an unfocused appearance. In nonpermeabilized samples (C), very few monocyte-associated E. coli are seen, presumably because the majority have been internalized and are not accessible to the external probe. The smaller, orange points, which lack the characteristic rod shape of E. coli, may be in part due to nonspecific staining. A control sample (D) in which naive WBC were permeabilized and stained shows only diffuse background staining and no distinct structures. Magnification, x1000.

 

View this table:
[in this window]
[in a new window]
  		Table III. Internalization of E. coli by human monocytes during the transfer reaction1

 
The question of bacterial killing was addressed by measuring CFU in samples incubated with and without WBC for 1 h at 37°C. Both WBC and E were lysed following the incubation, allowing all live E. coli, internal or external, to be counted (Table IVGo). In experiments without HP, WBC did show a limited ability to kill free E. coli; however, in all cases bacteria bound to E via HP were killed to a greater extent. Specific killing of approximately 1 log unit or more is evident over a wide range of E. coli to monocyte ratios. The killing capabilities of monocytes varied from donor to donor, but HP consistently augmented killing. To test whether the increased killing was the result of immune complex formation alone, killing was assayed for E. coli opsonized with mAb 44 and mixed with E and WBC (Table IVGo, Expt. 2). Killing in this system occurred at an intermediate level, between that of free E. coli and that of HP-opsonized E-bound E. coli, indicating that although specific (mAb 44) IgG binding facilitates bacterial killing, the E-HP system enhances it further.

Blockade of transfer and internalization

To determine which monocyte receptors might be instrumental in transfer, monocytes were preincubated with saturating amounts of certain mAbs to determine whether either removal of E. coli from E or uptake of E. coli by monocytes could be blocked. The results presented in Figure 7GoA indicate that mAbs respectively specific for FcRII (IV.3), CR1 (7G9), or an isotype-matched control for 7G9 (7B7) only weakly blocked monocyte-mediated removal of E. coli from E. However, these reagents were far more effective in blocking uptake by the monocytes of putatively released E. coli (Fig. 7GoB). This effect was most evident when a mixture of all three mAbs is used. Although removal of E. coli from the E was only weakly inhibited, uptake by the monocytes was substantially blocked in this case. For both removal from E and uptake by monocytes, anti-CR1 mAb 7G9 resulted in only slightly more blockade than its isotype control.



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 7. Inhibition of distinct steps of the transfer reaction: inhibition of removal of E. coli from E (A) and inhibition of uptake of E. coli by monocytes (B). WBC were preincubated with IV.3 (anti-FcRII), 7G9 (anti-CR1), 7B7 (irrelevant specificity, isotype control for 7G9), or a combination of all three mAbs for 4 h on ice or with a protease inhibitor mixture, EDTA, or EDTA plus Ca2+ for 30 min on ice. Control samples were preincubated in RPMI. E-HP-FITC-E. coli complexes were added, and transfer was allowed to occur for 5 min at 37°C. The cell populations were then separated, and E (A) and monocytes (B) were analyzed by FACS. Values represent the mean ± SD, with n = 4 for IV.3, 7B7, 7G9, and EDTA, and n = 2 for IV.3/7B7/7G9, protease inhibitor, and EDTA plus Ca2+.

 
We also sought to demonstrate directly the importance of a protease by using a mixture of protease inhibitors to block transfer and uptake (Fig. 7Go). The results indicate that if indeed a protease is involved in the transfer reaction, it is clearly not inhibited by the common protease inhibitors available. Incubation with EDTA only partially blocked release of E. coli from E, but it totally abrogated uptake of the bacteria by monocytes (Fig. 7Go). However, when Ca2+ was restored to the system, transfer resumed near control levels, suggesting that EDTA may inhibit Ca2+ signaling, rather than inhibiting a metalloprotease. To test for the secretion of a protease-like factor resistant to the inhibitors used, supernatants were collected from the monocyte/E mixture after 5 min of transfer. These supernatants were then added to suspensions of fresh E-HP-E. coli complexes and allowed to incubate for 5 min at 37°C. No release of E. coli occurred (results not shown).

To ascertain which monocyte cellular processes might be important in the removal of E. coli from E or the subsequent uptake and internalization of the bacteria by the phagocyte, the monocytes were incubated with a variety of metabolic inhibitors and then used in the transfer reaction (Table VGo). Only EDTA and cytochalasin D (an inhibitor of actin polymerization) (29) had significant inhibitory effects on the removal of bacteria from the E. However, use of genistein (a tyrosine kinase inhibitor) (30) and staurosporine (a protein kinase C inhibitor) (31) along with EDTA and cytochalasin D consistently led to lower numbers of E. coli being taken up and internalized by monocytes, as indicated by the decreases in both the percentage of positive cells and phagocytic indexes in the permeabilized samples (Table VGo). With EDTA and staurosporine, the reduced number of E. coli internalized appears to be simply a natural consequence of having fewer E. coli bound to the monocytes and available for internalization. The results for the use of cytochalasin D, and to a lesser extent genistein, are more complex. The relatively high level of positive cells in the nonpermeabilized samples suggest that these reagents may block internalization as well as uptake. Compared with the control samples in which approximately 95% of the E. coli associated with the monocytes were internalized, only 61% were internalized when incubated in the presence of genistein (95-37)/95 (based on phagocytic indices (Table VGo)), and cytochalasin completely blocked internalization. In the studies with cytochalasin D (Table VGo), the slightly higher number of E. coli (56) bound to the nonpermeabilized cells compared with the permeabilized cells (42) is probably due to a small error in counting. Colchecine (an inhibitor of microtubule assembly) (32) and sphingosine (another inhibitor of protein kinase C) (33) have no effect on transfer or internalization.


View this table:
[in this window]
[in a new window]
  		Table V. Inhibition of transfer and internalization by metabolic inhibitors1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HP have shown promise as potential therapeutic agents for the clearance of blood-borne pathogens from the circulation. In in vivo experiments, HP were able to clear from the circulation up to 6 log units of a circulating prototype innocuous target pathogen (11). Current work is focused on extending the HP approach to biologically relevant bacterial and viral pathogens. In the in vitro model presented herein, we demonstrate that specific HP-mediated E binding of E. coli occurs quite efficiently and rapidly (Table IGo and Fig. 1Go), and such binding can then promote the transfer ( Figs. 2–4GoGoGo and Table IIGo), internalization (Fig. 6Go and Table IIIGo), and killing (Table IVGo) of this bacterium by human monocytes. In this respect our findings reinforce and extend the original description of the biologic role of immune adherence in host defense first described by Nelson (1). However, as opposed to classic immune adherence, the reactions mediated by HP are accomplished in the absence of complement. The HP complex, rather than C3b, insures binding of the IC to E via CR1; once bound, the HP-Ag IC is recognized and processed by human monocytes. Our recent in vivo studies of HP-mediated clearance of target substrates also suggest that IC bound to E CR1 via HP are removed from the circulation by a mechanism quite similar to the process by which complement-opsonized immune complex substrates bound to E are cleared to the liver (8, 10, 11, 21).

Uptake of HP-opsonized E. coli by monocytes was greater than that observed when the bacteria were first bound to E via HP, washed, and then added to WBC (Fig. 4GoB). However, in the bloodstream the majority of CR1 is associated with E, and E CR1 competes quite effectively for C3b- or HP-opsonized substrates (3, 4, 5, 6, 7, 8, 9, 10, 11), so that HP-mediated binding precedes transfer to phagocytic cells. We anticipate that a similar pathway would be followed for HP-mediated clearance of bacteria under conditions in which the E are in very large excess over bacteria in the bloodstream.

With respect to both the in vitro and in vivo transfer reaction, there are several, presumably interrelated and possibly sequential, steps that must take place for the monocytes to destroy pathogens bound to E via HP. The HP-Ag complex must be released from the E, and then it must be bound, internalized, and degraded by the monocyte. These steps would in aggregate constitute the complete transfer reaction. Since no E sequestration or phagocytosis is seen in either the in vivo clearance studies (3, 4, 11, 21, 26) or in the present in vitro model, a critical step in transfer most likely involves monocyte-mediated release of the HP-Ag complex from E either before or shortly after it binds to the phagocyte. In previous in vivo clearance studies in monkeys using similar model complexes, we demonstrated that as IgG-containing IC were removed from E, there was a concerted loss in E CR1 (8, 21). In the present in vitro model system, a small (13%) but significant loss of CR1 from human E was demonstrable after transfer of E. coli (initially bound to the E via HP) to monocytes. Loss of CR1 has also been observed in conjunction with in vivo clearance of complement-opsonized IC in both human and primate models, and low CR1 levels are a characteristic of several diseases involving chronic immune complex processing (3, 27, 34, 35, 36, 37, 38, 39). Taken together, these findings support the hypothesis that CR1 is proteolytically cleaved during the course of transfer, releasing the CR1-HP-Ag complex and allowing it to be taken up by the phagocyte (8, 21, 27).

It should be noted that the situation is more complex in the in vitro model for monkey E. CR1 is lost during transfer of either HP-E. coli complexes or HP alone; we also found a smaller, but significant, loss in CR1 (Fig. 5Go) when naive E were incubated with human monocytes. Due to this background constitutive activity, there was a far greater net loss of E CR1 after transfer of the HP-E. coli complexes than was observed for human E. This activity could be due to a mismatch between the monkey E and human monocytes and will be examined in more detail in the future. However, in all cases loss of CR1 from monkey E after transfer of HP-bound E. coli was greater than loss of CR1 from naive monkey E, and the findings are therefore consistent with a proteolytic step during transfer. Finally, although we cannot definitely exclude a phospholipase in facilitating the release of monkey E CR1 (a GPI-linked membrane protein) during the transfer reaction, there is precedence for the proteolytic release of this class of protein. Huizinga et al. have reported that human neutrophil FcRIII (CD16), another GPI-linked membrane protein, is released from activated neutrophils due to the action of what is believed to be a membrane-associated protease (40).

The isolation and characterization of the putative monocyte-associated protease will be a focal point of future research. No inhibition of transfer was seen in the presence of a protease inhibitor mixture (Fig. 7Go), and supernatants collected from transfer mixtures did not release HP-bound E. coli from fresh E-HP-E. coli samples. These results suggest that proteolysis may occur in a solution-inaccessible pocket created by tight cell-cell interactions or that it may be mediated by a protease that is not susceptible to common protease inhibitors. Certain metalloproteases that mediate shedding of several surface proteins (including TNF-{alpha} and IL-6R) are similarly difficult to inhibit, responding only to specifically engineered protease inhibitors (41, 42). There is evidence for a similar proteolytic cleavage, in cells other than E, of specific membrane glycoproteins that share structural characteristics (the short consensus repeat motif) with CR1 (43, 44, 45). For example, Kazatchkine’s group (44) has reported that CR2 (CD21) on human lymphocytes appears to be cleaved in a domain proximal to the transmembrane portion of CD21, and this effect may be due to an endogenous enzyme activity that is also not blocked by common protease inhibitors.

The blocking experiments (Fig. 7Go) indicate that the role of monocyte receptors in distinct steps in the transfer reaction may be complex. Several different approaches led to the blockade of the majority of the net (complete) transfer to monocytes. However, there were marked quantitative differences in the effects of inhibition protocols on the loss of bacteria by the E compared with the uptake of bacteria by the monocytes. Whether inhibition was accomplished by performing the incubation on ice, blocking the monocyte receptors, or chelating divalent cations, removal of HP-Ag complexes from the E was inhibited to a lesser extent than monocyte uptake (Figs. 3Go and 7Go). That is, incubation with WBC led to robust release of substrate from E, and it was difficult to substantially inhibit this step. However, the same inhibition protocols proved to be far more effective in blocking uptake by the monocytes of the released complexes, which as a consequence of blockade were presumably released into the supernatant. These differences are consistent with the earlier CR1 loss data (Fig. 5Go) in implying that removal of substrate from E and subsequent monocyte uptake are not necessarily linked. Monocytes may have the ability to release HP-Ag complexes in a first step that may be independent of the second phase of the transfer reaction.

Binding and internalization of substrate by monocytes appear to take place in a concerted fashion; aside from the studies with cytochalasin D and genistein, virtually all monocyte-associated E. coli were found inside the cell, even after short incubations (Fig. 6Go and Table IIIGo). The receptors that facilitate these processes must be delineated. The findings in the inhibition studies (Fig. 7GoB and Table VGo) suggest that FcR participate in the binding and uptake of HP-Ag IC. On the other hand, in Emlen’s earlier in vitro IC transfer studies, he found monocyte CR1 to be the most important receptor in facilitating transfer of IC from E (46). This difference is probably attributable to differences in the nature of the substrates studied. The IC examined by Emlen were highly opsonized with complement (C3b), whereas the IC used in the present studies were formed in the absence of complement, but obviously may be able to bind to monocyte associated CR1 via the anti-CR1 mAbs in the HP (if they are not specifically engaged on E CR1). It is, therefore, likely that both FcR and CR1 play a role in the transfer reaction, with the influence of the individual receptors being to some extent proportional to the distribution of ligands on the transferred complex. Several groups have studied the differential requirements for CR- and FcR-mediated internalization and have found that the two have distinct mechanisms (23, 47). In inhibition studies, Newman demonstrated that while both CR1 and FcR require intact actin filaments and active protein kinase C for phagocytosis, only CR1-mediated internalization requires microtubule polymerization (23). Internalization in our in vitro model is consistent with FcR-mediated phagocytosis; it is sensitive to inhibitors of microfilament formation and tyrosine phosphorylation, the latter being a required signaling step in FcR-mediated internalization (48), but does not appear to require intact microtubules (Table VGo). The data using staurosporine and sphingosine to inhibit protein kinase C were inconclusive.

CR3 on monocytes is known to bind unopsonized E. coli via LPS in an EDTA-inhibitable and temperature-sensitive fashion (49). At 37°C in the absence of HP, E. coli appear to be only weakly taken up by monocytes (Fig. 3Go and Table IIGo), and therefore it seems unlikely that CR3 alone can facilitate the complete transfer reaction. However, EDTA partially blocks removal of E. coli from E and almost completely inhibits monocyte uptake of E. coli previously bound to E via HP (Table VGo and Fig. 7Go). These results suggests that CR3 may play a role in transfer, possibly cooperating with FcR in facilitating binding and phagocytosis of E. coli.

As noted above, the results suggest that monocytes express a protease that can cleave E CR1 when the cells are in direct contact. Such close and sustained contact between naive E and monocytes (or fixed tissue macrophages) would be highly unlikely in the bloodstream. In the liver, the close proximity of E containing bound IC and fixed tissue macrophages may facilitate specific FcR-ligand interactions between the macrophages and substrates bound to E CR1. These interactions could promote sustained contact and allow for the proteolysis of E CR1 and release and uptake of the bound substrates. We note that all studies reported herein were performed on freshly isolated, undifferentiated monocytes; whether the details of the transfer reaction to more differentiated cells are comparable remains to be established.

The results of these in vitro studies reinforce and extend earlier in vivo findings, demonstrating that pathogens bound to E CR1 via HP are rapidly transferred to phagocytes, and then they are internalized and destroyed. These experiments, therefore, provide additional evidence for the potential of HP as a therapy for diseases mediated by blood-borne pathogens.


   Acknowledgments
 
We thank Ms. Irina Dioumaeva for her assistance with the binding assays.


   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: E, erythrocytes; CR1, the primate E complement receptor; IC, immune complex; HP, heteropolymers; FcR, Fc receptor; WBC, white blood cells (mononuclear cells); MESF, molecules of equivalent soluble fluorochrome; GPI, glycophosphatidylinositol. Back

Received for publication August 15, 1997. Accepted for publication January 21, 1998.


   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. 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]
  3. Schifferli, J. A., R. P. Taylor. 1989. Physiological and pathological aspects of circulating immune complexes. Kidney Int. 35:993.[Medline]
  4. Cornacoff, J. B., L. A. Hebert, W. L. Smead, M. E. Van Aman, D. J. Birmingham, F. J. Waxman. 1983. Primate erythrocyte-immune complex-clearing mechanism. J. Clin. Invest. 71:236.
  5. Hebert, L. A., G. Cosio. 1987. The erythrocyte-immune complex-glomerulonephritis connection in man. Kidney Int. 31:877.[Medline]
  6. Taylor, R. P., W. M. Sutherland, C. Reist, D. J. Webb, E. L. Wright, R. H. Labugen. 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]
  7. Powers, J. H., B. L. Buster, C. J. Reist, E. Martin, M. Bridges, W. M. Sutherland, R. P. Taylor, W. M. Scheld. 1995. Complement-independent binding of microorganisms to primate erythrocytes in vitro by cross-linked monoclonal antibodies via complement receptor 1. Infect. Immun. 63:1329.[Abstract]
  8. 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]
  9. Taylor, R. P., P. J. Ferguson. 1995. Primate erythrocyte complement receptor (CR1) as an anchor site for bispecific-based therapies to clear pathogens or autoantibodies safely from the circulation. J. Hematother. 4:357.[Medline]
  10. Ferguson, P. J., E. N. Martin, K. L. Greene, S. Kuhn, D. S. Cafiso, G. Addona, R. P. Taylor. 1995. Antigen-based heteropolymers facilitate, via primate erythrocyte complement receptor type1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys. J. Immunol. 155:339.[Abstract]
  11. 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]
  12. Murphy, C. K., V. I. Kalve, P. E. Klebba. 1990. Surface topology of the Escherichia coli K-12 ferric enterobactin receptor. J. Bacteriol. 172:2736.[Abstract/Free Full Text]
  13. Rutz, J. M., J. Liu, J. A. Lyons, J. Goranson, S. K. Armstrong, M. A. McIntosh, J. B. Feix, P. E. Klebba. 1992. Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane. Science 258:471.[Abstract/Free Full Text]
  14. Jiang, X., M. A. Payne, Z. Cao, S. B. Foster, J. B. Feix, S. M. C. Newton, P. E. Klebba. 1997. Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria. Science 276:1261.[Abstract/Free Full Text]
  15. Tausk, F. A., J. 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.
  16. Segal, D. M., B. Bast. 1995. Production of bispecific antibodies. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology 2.13.1.. John Wiley and Sons,
  17. Oda, T., H. Maeda. 1986. A new simple fluorometric assay for phagocytosis. J. Immunol. Methods 88:175.[Medline]
  18. Muzykantov, V. R., R. P. Taylor. 1994. Attachment of biotinylated antibody to red blood cells: antigen-binding capacity of immunoerythrocytes and their susceptibility to lysis by complement. Anal. Biochem. 223:142.[Medline]
  19. Reinegel, 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]
  20. 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]
  21. 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]
  22. Mok, S. S., G. Evin, Q.-X. Lin, A. I. Smith, K. Beyreuther, C. L. Masters, D. H. Small. 1997. A novel metalloprotease in rat brain cleaves the amyloid precursor protein of Alzheimer’s disease generating amyloidogenic fragments. Biochemistry 36:156.[Medline]
  23. Newman, S. L., L. K. Mikus, M. A. Tucci. 1991. Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis. J. Immunol. 146:967.[Abstract]
  24. Wright, S. D., S. C. Silverstein. 1982. Tumor-producing phorbol esters stimulate C3b and C3b' receptor-mediated phagocytosis in cultured human monocytes. J. Exp. Med. 156:1149.[Abstract/Free Full Text]
  25. Kobayashi, K., K. Takahashi, S. Nagasawa. 1995. The role of tyrosine phosphorylation and Ca2+ accumulation in receptor-mediated phagocytosis of human neutrophils. J. Biochem. 117:1156.[Abstract/Free Full Text]
  26. Kimberly, R. P., J. C. Edberg, L. T. Merriam, S. B. Clarkson, J. C. Unkeless, R. P. Taylor. 1989. In vivo handling of soluble complement fixing Ab/dsDNA immune complexes in chimpanzees. J. Clin. Invest. 84:962.
  27. Ross, G. D., W. J. Yount, M. J. Walport, J. B. Winfield, C. J. Parker, C. R. Fuller, R. P. Taylor, B. L. Myones, P. J. Lachmann. 1985. 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.[Abstract]
  28. Birmingham, D. J., C. M. Logar, X.-P. Shen, W. Chen. 1996. The baboon erythrocyte complement receptor is a glycophosphatidylinositol-linked protein encoded by a homologue of the human CR1-like genetic element. J. Immunol. 157:2586.[Abstract]
  29. Cooper, J. A.. 1987. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105:1473.[Free Full Text]
  30. Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukamki. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262:5592.[Abstract/Free Full Text]
  31. Tamaoki, T., H. Namoto, I. Takahashi, Y. Kato, M. Morimoto, F. Tomita. 1986. Staurosporine, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135:397.[Medline]
  32. Wilson, L., J. R. Bamburg, S. B. Mizel, L. M. Grisham, K. M. Creswell. 1974. Interaction of drugs with microtubule proteins. Fed. Proc. 33:158.[Medline]
  33. Hannun, Y. A., C. R. Loomis, A. H. Merril, R. M. Bell. 1986. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J. Biol. Chem. 261:12604.[Abstract/Free Full Text]
  34. Miyakawa, Y., A. Yamada, K. Kosaka, F. Tsuda, E. Kosugi, M. Mayumi. 1981. Defective immune-adherence (C3b) receptor on erythrocytes from patients with systemic lupus erythematosus. Lancet ii:493.
  35. Walport, M. J., P. J. Lachmann. 1988. Erythrocyte complement receptor type 1, immune complexes, and the rheumatic diseases. Arthritis Rheum. 31:153.[Medline]
  36. Birmingham, D. J., L. A. Hebert, F. G. Cosio, M. E. van Aman. 1990. Immune complex erythrocyte complement receptor interactions in vivo during induction of glomerulonephritis. J. Lab. Clin. Med. 116:242.[Medline]
  37. Cosio, F. G., S. Xiao-Ping, 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]
  38. 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]
  39. Davies, K. A., A. M. Peters, H. L. C. Benyon, M. Walport. 1992. Immune complex processing in patients with systemic lupus erythematosus. J. Clin. Invest. 90:2075.
  40. Huizinga, T. W. J., M. de Haas, M. Kleijer, J. H. Nuijens, D. Roos, A. E. G. Kr. Von dem Borne. 1990. Soluble Fc{gamma} receptor III in human plasma originates from release by neutrophils. J. Clin. Invest. 86:416.
  41. Mohler, K. M., P. R. Sleath, J. F. Fitzner, D. P. Cerretti, M. Alderson, S. S. Kerwar, D. S. Torrance, C. Otto-Evans, T. Greenstreet, K. Weerawarna, S. R. Kronheim, M. Peterson, M. Gerhart, C. J. Kozlosky, C. J. Marsh, R. A. Black. 1994. Protection against a lethal dose of endotoxin by an inhibitor of tumor necrosis factor processing. Nature 370:218.[Medline]
  42. Mullberg, J., F. H. Durie, C. Otto-Evans, M. Alderson, S. Rose-John, D. Cosman, R. A. Black, K. M. Mohler. 1995. A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J. Immunol. 155:5198.[Abstract]
  43. 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]
  44. Fremeaux-Bacchi, V., I. Bernard, F. Maillet, J.-C. Mani, M. Fontaine, J.-V. 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]
  45. Preece, G., G. Murphy, and A. Ager. Metalloproteinase-mediated regulation of L-selectin levels on leucocytes. J. Biol. Chem. 271:11634.
  46. 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]
  47. Allen, L.-A. H., A. Aderem. 1996. Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J. Exp. Med. 184:627.[Abstract/Free Full Text]
  48. Greenberg, S., P. Chang, S. C. Silverstein. 1993. Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 177:529.[Abstract/Free Full Text]
  49. Wright, S. D., M. T. C. Jong. 1986. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. J. Exp. Med. 164:1876.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
X. Li, K. Su, C. Ji, A. J. Szalai, J. Wu, Y. Zhang, T. Zhou, R. P. Kimberly, and J. C. Edberg
Immune Opsonins Modulate BLyS/BAFF Release in a Receptor-Specific Fashion
J. Immunol., July 15, 2008; 181(2): 1012 - 1018.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. W. J. van de Sande, A. Fahal, H. Verbrugh, and A. van Belkum
Polymorphisms in Genes Involved in Innate Immunity Predispose Toward Mycetoma Susceptibility
J. Immunol., September 1, 2007; 179(5): 3065 - 3074.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. D. Kennedy, P. V. Beum, M. D. Solga, D. J. DiLillo, M. A. Lindorfer, C. E. Hess, J. J. Densmore, M. E. Williams, and R. P. Taylor
Rituximab Infusion Promotes Rapid Complement Depletion and Acute CD20 Loss in Chronic Lymphocytic Leukemia
J. Immunol., March 1, 2004; 172(5): 3280 - 3288.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. A. Lindorfer, A. Nardin, P. L. Foley, M. D. Solga, A. J. Bankovich, E. N. Martin, A. L. Henderson, C. W. Price, E. Gyimesi, C. P. Wozencraft, et al.
Targeting of Pseudomonas aeruginosa in the Bloodstream with Bispecific Monoclonal Antibodies
J. Immunol., August 15, 2001; 167(4): 2240 - 2249.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
V. Le Cabec, C. Cols, and I. Maridonneau-Parini
Nonopsonic Phagocytosis of Zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) Involves Distinct Molecular Determinants and Is or Is Not Coupled with NADPH Oxidase Activation
Infect. Immun., August 1, 2000; 68(8): 4736 - 4745.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. L. Reinagel and R. P. Taylor
Transfer of Immune Complexes from Erythrocyte CR1 to Mouse Macrophages
J. Immunol., February 15, 2000; 164(4): 1977 - 1985.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuhn, S. E.
Right arrow Articles by Taylor, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuhn, S. E.
Right arrow Articles by Taylor, R. P.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CYTOCHALASIN D
*ETHYLENEDIAMINE TETRAACETIC ACID

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS