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


     
 

The Journal of Immunology, 2007, 179, 5543 -5552
Copyright © 2007 by The American Association of Immunologists, Inc.
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 Google Scholar
Google Scholar
Right arrow Articles by Pawluczkowycz, A. W.
Right arrow Articles by Taylor, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pawluczkowycz, A. W.
Right arrow Articles by Taylor, R. P.

Hematin Promotes Complement Alternative Pathway-Mediated Deposition of C3 Activation Fragments on Human Erythrocytes: Potential Implications for the Pathogenesis of Anemia in Malaria1

Andrew W. Pawluczkowycz*, Margaret A. Lindorfer*, John N. Waitumbi{dagger} and Ronald P. Taylor2,*

* Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA 22908; and {dagger} Walter Reed Project and Kenya Medical Research Institute, Kisumu, Kenya


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Childhood malaria caused by Plasmodium falciparum is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported, based on clinical observations and in vitro models, that complement control proteins on erythrocytes such as CR1, the immune adherence receptor specific for C3b, may be reduced in childhood malaria, suggesting a possible role for complement in erythrocyte destruction. Intravascular lysis of iE by P. falciparum leads to release of erythrocyte breakdown products such as hemoglobin and hematin, which have inflammatory properties. In the present article, we demonstrate that in serum and in anticoagulated whole blood, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on erythrocytes. The degree of C3 fragment deposition is directly correlated with erythrocyte CR1 levels, and erythrocytes opsonized with large amounts of C3dg form rosettes with Raji cells, which express CR2, the C3dg receptor which is expressed on several types of B cells in the spleen. Thus, the reaction mediated by hematin promotes opsonization and possible clearance of the youngest (highest CR1) erythrocytes. A mAb specific for C3b, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this mAb in nonhuman primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of targeted therapies based on inhibiting the alternative pathway of complement.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intravascular lysis of erythrocytes is associated with a number of pathologic conditions and can lead to release into the bloodstream of substantial amounts of hemoglobin and its breakdown products, including hematin (Fe III protoporphyrin IX) (1, 2, 3, 4). Both free hemoglobin and hematin can promote severe tissue injury at a variety of sites, and several mechanisms serve to protect the body from these inflammatory agents (2, 3, 4, 5, 6, 7, 8). Under quiescent conditions, hemoglobin or hematin released into the circulation are rapidly sequestered by haptoglobin and hemopexin, respectively, and then cleared by the liver, thus preventing the buildup of these toxic agents in the blood (3, 7). However, under pathologic conditions in diseases such as paroxysmal nocturnal hemoglobinuria or malaria, these defense mechanisms may be overwhelmed by the release of large amounts of erythrocyte breakdown products, and due to the failure to neutralize these agents, secondary pathologies may ensue (2, 3, 4, 9). Malaria-associated anemia in young children is further complicated because, in many cases, the number of erythrocytes that are lost far exceeds the number of erythrocytes that are actually infected by the Plasmodium falciparum (Pf)3 parasite (10, 11, 12, 13, 14, 15, 16). It is likely that for every one Pf-infected erythrocyte (iE) that is destroyed, 10 or more uninfected erythrocytes (uE) are cleared.

Although the mechanism(s) responsible for clearance of uE in malaria have not been delineated, several theories have been proposed (10, 11, 13, 14, 15, 16, 17, 18, 19, 20). Hematin binds to erythrocytes and destabilizes the erythrocyte membrane, and in fact hematin bound to erythrocytes can promote oxidative damage, thus suggesting a potential mechanism for uE destruction (5, 6, 21). Alternatively, modification of the erythrocyte surface by bound IgG and/or parasite-derived Ags may lead to reduced erythrocyte deformability, which has also been implicated in uE loss (10, 11, 13). Other theories based on rodent models have suggested that Pf infection induces an inflammatory condition in the bloodstream that leads to enhanced and rapid clearance of both iE and uE (15).

Our laboratories have investigated the role of complement in the pathogenesis of malarial anemia. During childhood malarial anemia, the erythrocytes remaining in the circulation have reduced levels of two key complement proteins: CR1, the immune adherence receptor for C3b, and CD55, a complement control protein that down-regulates deposition of C3b on cells (12, 20, 22). Moreover, several reports, based on in vitro investigations and clinical observations, have revealed that complement-activation products, in particular C3b and its fragments, can be found on erythrocytes in the bloodstream during many phases of malaria infections (23, 24, 25), and our in vitro studies have demonstrated loss of CD55 upon chelation with anti-CD55 mAbs followed by interaction with model monocyte/macrophages (26). In view of the fact that free hematin can activate complement (27), and because hematin is known to bind to erythrocytes (5, 6, 21), we have examined the hypothesis that activation of complement, promoted by the binding of hematin to uE in the bloodstream, may play an important role in the pathogenesis of malarial anemia and the destruction of uE.

In this study, we present data demonstrating that in the presence of normal human serum (NHS) or in whole blood, hematin activates the alternative pathway (AP) of complement and promotes rapid deposition of complement C3 activation products C3b, iC3b, and C3dg on erythrocytes. CR1 on the erythrocytes appears to play a key role in promoting this deposition, and we find that repeated additions of fresh NHS and hematin to previously reacted erythrocytes continue to promote deposition of additional complement C3 fragments on erythrocytes in substantial excess of the total number of CR1. After multiple cycles of treatment with NHS and hematin, the treated erythrocytes form rosettes when mixed with Raji cells, which express CD21, the receptor for C3dg (28). Our findings suggest a possible new mechanism by which hematin, released into the bloodstream after lysis of Pf-iE, may, along with complement, promote the clearance and destruction of uE in malaria.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
mAbs, reagents, and cells

mAbs 7C12, 8E11, 3E7, and 2C5 (all specific for C3b/iC3b) and mAb 1H8 (specific for C3b/iC3b/C3dg) have been previously described (29, 30, 31, 32). mAbs 1E6 and 3C11, both specific for C3b/iC3b, and mAb 14A10, specific for C3b/iC3b/C3dg, were prepared and characterized following published procedures (30, 32). In this panel, mAb 14A10 and mAb 1H8 compete for binding to the same epitope on C3dg, and mAb 2C5 and mAb 3E7 compete for a different epitope on the C3c portion of C3b/iC3b. Otherwise, the mAbs all bind to separate sites on C3 fragments. mAbs 1B4, HB8592, 3D9, and 7G9, all specific for CR1, have been previously described (33, 34, 35, 36). Abs were labeled with Alexa (Al) 488 and Al647 according to the manufacturer’s directions (Molecular Probes). The following reagents were obtained commercially, as indicated: Al488 goat anti-mouse IgG and Al647 rat anti-mouse IgG (Molecular Probes); FITC rat anti-mouse C3 Ab (Cedarlane Laboratories); PerCP-streptavidin (BD Biosciences); hematin, papain, trypsin, and zymosan A (Sigma-Aldrich). Zymosan was prepared by dispersion at 8 mg/ml in PBS and immersion in a boiling water bath for 10 min (32). NHS was obtained from volunteers following written consent, and in all experiments, erythrocyte blood types were matched with sera. Panhematin, freshly prepared, was obtained from the University of Virginia Hospital Pharmacy. Flow cytometry was accomplished on a FACSCalibur flow cytometer (BD Biosciences), and mean fluorescence intensities were converted to molecules of equivalent soluble fluorochrome (MESF) using calibrated standard beads (Spherotech) (31). The B lymphoblastoid Raji cell line (American Type Culture Collection) was cultured in RPMI 1640 medium supplemented with penicillin and streptomycin (Invitrogen Life Technologies). Complement proteins and factor B-depleted sera were obtained from Complement Technology.

Hematin reaction with erythrocytes

Hematin was dissolved in 0.1 M NaOH and titrated with 0.1 M HCl until the pH measured 8.0. The hematin concentration was determined by diluting aliquots in 20 mM NaOH/2% SDS solutions and measuring the OD at 400 nm (37). Erythrocytes were isolated from whole blood anticoagulated with EDTA (38) and pelleted; the buffy coat was removed and the erythrocytes were washed three times with either gelatin-veronal buffer (GVB) supplemented with 5 mM MgCl2/8 mM EGTA (AP buffer) or with 0.5 mM MgCl2/0.1 mM CaCl2 (CP buffer). NHS was also supplemented with either mixture of Mg-EGTA (AP) or Ca/Mg (CP) and thus all experiments are operationally defined as either "AP" or "CP", although both pathways can proceed in CP buffer.

Hematin-mediated complement activation and C3 fragment deposition were studied by combining erythrocytes, NHS, and hematin, and incubating the mixtures at 37°C for varying times. The final erythrocyte hematocrit (% hct), percentage NHS, and hematin concentration are noted in the figures and tables. Erythrocytes from >10 different normal donors were examined in this study. In some experiments, hematin was combined with erythrocytes and human factor B-depleted NHS in AP or CP buffer and incubated at 37°C for 15 min, in the presence or absence of added factor B. When multiple cycles of opsonization were performed, the reactions were stopped with the addition of four volumes of cold buffer solution, centrifuged, the supernatant was removed, and the opsonization repeated. In all experiments, after the final incubation, erythrocytes were washed five times with BSA-PBS and then probed with selected mAbs for 30 min at room temperature, washed three times with BSA-PBS, resuspended in 1% paraformaldehyde, and the samples were immediately analyzed by flow cytometry.

Alternatively, erythrocytes (10% hct) were first reacted with papain or trypsin (100 µg/ml) to remove CR1 (39, 40). After treatment at 37°C for up to 1 h, the erythrocytes were washed extensively and then subjected to repeated cycles of hematin/NHS treatment, followed by probing and analysis by flow cytometry. In some experiments, the protease-treated erythrocytes were first mixed with FITC-labeled untreated erythrocytes to determine whether activation of complement on these naive FITC-labeled erythrocytes might promote C3b deposition on the erythrocytes in which CR1 had been removed.

Activation of the AP of complement by hematin

The ability of hematin to suppress the AP-mediated deposition of C3 fragments on zymosan (32) was studied by first incubating NHS, in AP buffer, with hematin before the addition of zymosan. Mixtures of NHS and hematin were combined with zymosan at differing times, and after incubation, the zymosan was washed and probed with mAbs specific for C3 fragments and analyzed by flow cytometry.

Rosette formation

Rosette formation using Raji cells was conducted by first treating erythrocytes with multiple cycles of NHS and hematin as described above. Treated or untreated erythrocytes were mixed with Raji cells, briefly centrifuged, and incubated at 37°C for 15 min. Aliquots were removed and examined by light microscopy. At least 100 Raji cells were counted, and rosette formation was defined as Raji cells juxtaposed to at least five erythrocytes.

Western blots and immunoprecipitation

Erythrocytes were reacted with either NHS or with NHS plus hematin (300 µg/ml) for seven consecutive cycles; after three washes, the pelleted erythrocytes were lysed in 40 volumes of 0.005 M sodium phosphate (pH 7.4) in the presence of protease inhibitors (41). The membranes were then collected by centrifugation at 30,000 x g and solubilized in PBS with 1% Nonidet P-40, and frozen for later analysis. The protein concentrations in the membrane preparations were determined with the BCA reagent (Pierce). Alternatively, the treated erythrocytes were washed and directly lysed in radioimmunoprecipitation (RIPA) buffer (42) and then subjected to immunoprecipitation as follows. The lysates were first reacted with mouse IgG (100 µg/750 µl of lysate) and with protein A-Sepharose (Sigma-Aldrich) for 30 min at 4°C. After a clearing spin for 1 min at 200 x g, the supernatants were then reacted for 2 h with anti-C3b/iC3b/C3dg mAb 1H8 followed by additional protein A-Sepharose. Following an overnight incubation at 4°C, the mixtures were pelleted, and after four washes with PBS, followed by one wash with RIPA buffer, bound material was released by addition of 1 volume of 2x SDS loading buffer followed by boiling for 5 min. Both the solubilized membrane preparations and the material isolated by immunoprecipitation were subjected to SDS-PAGE on 4–15% gradient gels, followed by transfer to polyvinylidene difluoride membranes. Development was accomplished by addition of biotinylated mAbs (1 µg/ml) followed by SA-HRP (1/1000) (Pierce) and then reaction with ECL reagent (Amersham).

Graphical analysis and line fitting (see Fig. 5C) was performed using SigmaPlot 9.0 (SSI).


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 5. Dot plots demonstrate a correlation between erythrocyte CR1 levels and the number of C3 fragments deposited on erythrocytes (40% hct) after reaction with 300 µg/ml hematin in 40% NHS (three cycles of 15 min, CP). After C3 fragment deposition, the washed erythrocytes were simultaneously probed for CR1 (biotinylated anti-CR1 mAbs 7G9 and HB8592, followed by streptavidin-PerCP) and for deposited C3 fragments (FITC goat anti-human C3). A, Donor with relatively low CR1. The eight different colors separate the erythrocytes into eight populations, defined by CR1 levels. Conversion of channel numbers to MESF gives a mean MESF for erythrocyte CR1 = 1083. B, Similar analysis for a donor with higher erythrocyte CR1 gives a mean MESF = 2052. C, Summary of dot plots for six donors, in which C3 fragment deposition values, defined by MESF, are plotted against erythrocyte CR1 MESF values for each donor’s eight subpopulations. Thus, for each donor, eight data points (of a single color) are displayed. The pink inverted triangles represent erythrocytes for the donor in A, and the purple circles represent erythrocytes for the donor in B. The data points can be reasonably described by two intersecting straight lines. It appears that above ~1500 MESF units of CR1, there is an approximate linear correlation between CR1 values and C3 fragment deposition. The equations for the two lines (lower and upper, respectively) where y is the FITC MESF signal and x is the PerCP MESF signal are: y = 3.84x + 341; and y = 6.92x – 4286.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Hematin promotes deposition of C3 fragments on erythrocytes in the presence of NHS

Addition of hematin to erythrocytes in NHS-AP buffer leads to deposition of C3 fragments on the erythrocytes, and the concentration dependence of the dose response suggests that a threshold of hematin concentration (>100 µg/ml) is needed for substantial amounts of C3 fragments to be deposited (Fig. 1A). Fig. 1B reveals that NHS concentrations as low as 15% are adequate to support C3 deposition. To model the potential effects of repeated cycles of destruction of iE and release of hematin in malaria, we examined the effect of multiple treatments of erythrocytes with fresh NHS and hematin on C3 fragment deposition. Fig. 1C demonstrates that each additional cycle of exposure of human erythrocytes to fresh NHS and hematin increases the number of deposited C3 fragments. Control experiments, in which erythrocytes were treated with NHS alone (up to 10 cycles) gave much less C3 fragment deposition. For example, in one experiment, after 10 cycles of treatment of erythrocytes with NHS alone, the signal for mAb 1H8 binding was 401 MESF, compared with 1400 MESF for erythrocytes treated with serum and hematin. The background MESF signal for naive erythrocytes (not reacted with mAb 1H8) was 213 MESF. Hematin-mediated deposition of C3 fragments was demonstrable with a panel of mAbs (Fig. 1D), specific for either C3b/iC3b/C3dg (mAbs 14A10 and 1H8) or specific for only C3b/iC3b (mAbs 7C12, 3C11, 2C5, 8E11, and 3E7). We found that mAbs 1H8 and 14A10 tended to give the largest signals after 10 or more cycles of treatment, presumably because much of the C3b/iC3b had been degraded to C3dg, thus favoring these mAbs, which recognize C3dg as well as C3b and iC3b. Isotype control mAbs showed no binding to erythrocytes treated with either NHS or NHS plus hematin (not shown). Recovery of erythrocytes after 5–10 cycles of treatment with hematin plus serum was >90% of the recovery observed for samples reacted with serum alone, suggesting there may have been a small amount of erythrocyte lysis during reaction with hematin.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 1. Hematin mediates deposition of C3 fragments on human erythrocytes in the presence of NHS under a variety of conditions. A, Erythrocytes (5% hct) in 50% NHS in AP buffer, were mixed with varying concentrations of hematin and incubated at 37°C for 15 min, washed, and probed with Al488 anti-C3b/iC3b/C3dg mAb 1H8 and then analyzed by flow cytometry. The dashed line shows that C3 fragment deposition is abrogated in comparable experiments in NHS-EDTA in the presence or absence of 200 µg/ml hematin. In this and other experiments, unless otherwise specified, all points are in triplicate; means and SD are shown. In many cases, the error bars are smaller than the symbols. B, Effects of NHS concentration in AP buffer on the C3 fragment deposition reaction. Similar conditions as in A: 40% hct and 200 µg/ml hematin. C, Multiple 15-min cycles of incubation of erythrocytes (5% hct) with 300 µg/ml hematin and 50% NHS in AP buffer allow for increased accumulation of C3 fragments on erythrocytes. D, Hematin-mediated deposition of C3 fragments on erythrocytes is demonstrable using a variety of mAbs specific for C3 fragments. Erythrocytes (40% hct) were reacted in 40% NHS in CP buffer, ± 300 µg/ml hematin for a total of four cycles, and then probed with the indicated mAbs (all single determinations). E, Both hematin and panhematin (300 µg/ml in each case) promote C3 fragment deposition on erythrocytes (40% hct) in 40% NHS in CP buffer (four cycles).

 
To further generalize our findings, we extended the assays of C3 fragment deposition to include tests with freshly prepared panhematin, a drug used in the treatment of porphyria. In these experiments, we used FITC-labeled goat-anti-human C3 Abs as probes. Again, compared with findings with NHS alone, we observed substantial C3 fragment deposition for erythrocytes treated with either form of hematin, both examined at 300 µg/ml (Fig. 1E).

Hematin effectively activates the AP of complement

We followed AP complement consumption by hematin by first preincubating hematin with NHS. The reacted NHS was then combined with zymosan, and C3 fragment deposition on this AP-activating substrate was measured. Table I shows that addition of NHS to zymosan promotes substantial deposition of C3 fragments on the zymosan, as revealed with two different C3dg-specific mAb probes. However, preincubation of NHS with hematin rapidly consumes the AP of complement; deposition of C3 fragments on the zymosan is almost completely inhibited after a 2-min preincubation of NHS with hematin. Moreover, even when the two substrates are first mixed and then reacted with NHS, it is clear that the hematin effectively competes for complement, because deposition of C3 fragments on the zymosan decreases substantially from 1.5 million MESF to only 8130 MESF (mAb 1H8 as probe). Similar results were obtained when mAb 14A10 was used to assay for C3 fragment deposition. We further investigated hematin activation of the AP of complement by using erythrocyte-hematin mixtures as substrates. After a single incubation of erythrocytes with NHS and hematin, C3 fragment deposition corresponds to ~650 MESF (Table II). In analogy to the experiment using zymosan, we find that preincubation of NHS with hematin alone severely decreases subsequent C3 fragment deposition on erythrocytes in hematin-erythrocyte mixtures; C3 fragment deposition is reduced to background levels (~320 MESF), indicating that the AP was consumed during the incubation of NHS with hematin. We note that although the MESF signal for NHS-opsonized zymosan (>1 million MESF) is much greater than the signal for NHS-opsonized hematin-erythrocyte mixtures (650 MESF), the two experiments illustrated in Tables I and II, respectively, both demonstrate that brief incubation of hematin with NHS alone consumes the AP very effectively.


View this table:
[in this window]
[in a new window]

  		Table I. Pretreatment of NHS with hematin consumes the AP of complement and blocks deposition of C3 fragments on two substrates

 

View this table:
[in this window]
[in a new window]

  		Table II. Analysis of C3 fragment deposition on hematin-erythrocyte mixtures

 
Hematin-mediated deposition of C3 fragments on erythrocytes occurs in whole blood, requires factor B, and is blocked by mAb 3E7

To gain additional insight as to mechanism, and to model conditions closer to those found in the circulation of a patient with malaria, we studied hematin-mediated deposition of C3 fragments on erythrocytes in whole blood anticoagulated with lepirudin, an agent that selectively inhibits coagulation while leaving complement untouched (43). We compared this condition to one in which washed erythrocytes were reconstituted in normal human plasma obtained from blood anticoagulated with lepirudin. Fig. 2A reveals that under both conditions, C3 fragments are deposited on erythrocytes at hematin concentrations >100 µg/ml. Fewer C3 fragments are deposited on erythrocytes in whole blood at intermediate concentrations of hematin, but it is noteworthy that similar levels of C3 fragment deposition are observed at hematin levels of 300 µg/ml or more. Our findings suggest that the hematin-mediated C3 fragment deposition reaction is promoted through the AP pathway of complement. The results illustrated in Fig. 2B demonstrate that in both AP buffer and in CP buffer, similar patterns of C3 fragment deposition are evident, which is reasonable as both buffers are permissive for the AP. To further examine the hypothesis that C3 fragment deposition is mediated by the AP, we tested factor B-depleted serum and found that hematin could not promote C3 fragment deposition on erythrocytes in this serum (Fig. 2C). To restore C3 fragment deposition, both factor B and hematin must be added to this depleted serum, and under these conditions we observe hematin-mediated deposition of C3 fragments in both CP and AP buffers. We have previously reported that mAb 3E7, which binds to C3b/iC3b, enhances the action of the CP and completely blocks the AP of complement, by binding to C3b-opsonized substrates in a manner which blocks binding of both factor B and factor H to C3b (32). In the experiment illustrated in Fig. 2D, erythrocytes were exposed to NHS and hematin in a 10-cycle experiment, in the presence and absence of mAb 3E7 for each cycle. The results indicate that mAb 3E7 completely suppresses C3 fragment deposition on the erythrocytes, providing additional evidence that the reaction proceeds via the AP of complement.


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 2. Hematin mediates deposition of C3 fragments on erythrocytes (E) in anticoagulated whole blood, and several independent experiments suggest that the reaction proceeds via the AP. A, Blood was collected in the presence of lepirudin (final concentration of 25 µg/ml) and then varying amounts of hematin were added and deposition of C3 fragments on the erythrocytes measured after three cycles of treatment. In each succeeding cycle, fresh lepirudin-containing plasma and hematin were added. As a control, washed erythrocytes from the same normal donor, reconstituted to 50% hct in autologous lepirudin-containing plasma, were reacted with comparable amounts of hematin. B, Comparable amounts of C3 fragment deposition are evident after reacting erythrocytes (40% hct) with 40% NHS and hematin (300 µg/ml, three cycles) in either CP or AP buffer. C, Erythrocytes (40% hct) from the same donor used in the experiment illustrated in B were reacted with 40% factor B-depleted NHS, in the presence and absence of 300 µg/ml hematin, ± added factor B (20 µg/ml final concentration). Substantial hematin-promoted deposition of C3 fragments (three cycles) occurs in both CP and AP buffer, when the serum is supplemented with factor B. D, Addition of mAb 3E7 (200 µg/ml) to 45% NHS completely blocks hematin-mediated deposition of C3 fragments on human erythrocytes (5% hct, 10 cycles, AP buffer, 270 µg/ml hematin). mAb 1H8, used for analysis, binds to C3 fragments at a site distinct from that occupied by mAb 3E7 (32 ).

 
On the role of erythrocyte CR1 in the C3 fragment deposition reaction

C3b is the natural ligand for CR1 (44, 45), and therefore we investigated the possible role of CR1 in the hematin-mediated deposition reaction. Erythrocytes were treated with proteases (trypsin or papain) under conditions previously established to remove almost all CR1 from the erythrocytes (39, 40) and then the erythrocytes were reacted with NHS and hematin. The erythrocytes were examined by probing with mAbs specific for either CR1 or for C3 fragments, and secondarily developed with Al488 goat anti-mouse IgG. The results, illustrated in Fig. 3A, reveal that papain treatment of the erythrocytes does indeed mediate loss of erythrocyte CR1, and coincident with this loss, the hematin-mediated deposition of C3 fragments on the erythrocytes is almost completely abrogated. Similarly, treatment of the erythrocytes with trypsin greatly reduced CR1 levels (data not shown) and trypsin treatment also reduced C3 fragment deposition (Fig. 3B). To determine whether C3 deposition on the trypsin-treated erythrocytes could be mediated by a trans effect, we mixed naive, FITC-labeled erythrocytes with trypsin-treated erythrocytes and then measured C3 fragment deposition on both erythrocyte populations in this mixture, in the presence and absence of hematin. The results in Fig. 3C indicate that even in the presence of naive erythrocytes in which C3 fragment deposition is clearly demonstrable, far less deposition occurs on the trypsin-treated erythrocytes.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 3. Treatment of erythrocytes with proteases which remove CR1 also substantially decreases the ability of hematin to promote deposition of C3 fragments on erythrocytes. A, After reaction of erythrocytes with 100 µg/ml papain for varying time periods, the erythrocytes (5% hct) were washed and reacted with 300 µg/ml hematin in 45% NHS (single cycle, CP) and then assayed for either CR1 (mAb 7G9) or for bound C3 fragments (mAb 1H8). Binding of the mAbs was revealed by secondary development with Al488 goat anti-mouse IgG. B and C, Erythrocytes were treated with trypsin to remove CR1, and then these erythrocytes or FITC-labeled naive erythrocytes were reacted with 40% NHS ± 300 µg/ml hematin for five cycles (CP). In B, the two erythrocyte populations were separately reacted with NHS and hematin, while in C, they were mixed before reaction with NHS and hematin. After five cycles, the erythrocytes were washed and probed with Al633 mAb 1H8. In C, analysis by flow cytometry allowed for separate identification of the two erythrocyte populations: naive, FITC-labeled erythrocytes or trypsin-treated but otherwise unlabeled erythrocytes. D, After five cycles of reaction of erythrocytes (40% hct) with 300 µg/ml hematin and 40% NHS (AP), the number of C3 fragments deposited on the erythrocytes is considerably greater than the number of erythrocyte CR1, which remains constant throughout the reaction. Comparisons are based on probing with mAb 1H8, specific for C3 fragments, or with mAb 1B4, specific for CR1, followed by development with Al647 rat anti-mouse IgG.

 
To further assess the quantitative aspects of the requirements for CR1, erythrocytes were subjected to multiple cycles of reaction with fresh NHS and hematin. After either one or five cycles of treatment, the erythrocytes were probed with mAb 1H8, specific for C3dg, or with mAb 1B4, specific for CR1, followed in each case by Al647 rat anti-mouse IgG. The results (Fig. 3D) indicate that even after five treatments of erythrocytes with hematin and NHS, binding of the anti-CR1 mAb 1B4 remains constant, and thus the deposited C3 fragments do not block binding of this anti-CR1 mAb. After five cycles of treatment, the amount of anti-C3dg mAb 1H8 that binds to the cells is in great excess over the amount of anti-CR1 mAb 1B4 that binds, suggesting that CR1 is able to mediate C3 fragment deposition in excess of the total number of CR1 molecules on the erythrocytes (Fig. 3D). Finally, it is well-established that anti-CR1 mAbs 1B4 and 3D9 cause C3b-opsonized immune complexes to dissociate from erythrocyte CR1 (33, 35, 46, 47). However, in contradistinction to these observations, addition of anti-CR1-blocking mAbs 1B4 or 3D9 to washed erythrocytes containing C3 deposition fragments does not cause these fragments to dissociate from the erythrocytes. In a separate experiment in which a single cycle of opsonization was used, C3 fragment binding corresponded to 613 ± 14 MESF units (n = 3, background of 300 MESF units). After addition of saturating amounts of either 1B4 or 3D9 to these opsonized cells, followed by an incubation of 30 min at 37°C, C3 fragment binding was unchanged; 602 ± 15 and 603 ± 14 MESF units, respectively (n = 3). This finding suggests that the C3 fragments are covalently bound to the erythrocytes.

Western blots confirm that hematin promotes covalent binding of C3 fragments to erythrocytes

We also used Western blotting and immunoprecipitation to determine whether reaction of erythrocytes with hematin in NHS promotes covalent deposition of C3 fragments on the erythrocytes. As illustrated in Fig. 4A, analysis of solubilized erythrocyte membranes from erythrocytes reacted with hematin plus NHS (treated), compared with membranes isolated after reaction with NHS alone (untreated), reveals two distinct bands based on probing with anti-C3b/iC3b/C3dg mAb 1H8; the first band is centered at a molecular mass of ~120 kDa and the second broader band identifies C3dg-positive fragments associated with proteins at molecular mass >200 kDa. The band at 120 kDa likely represents C3dg covalently bound to one or more unidentified erythrocyte membrane proteins, because the band does not stain with mAb 1E6, specific for C3b/iC3b (Fig. 4B). Also of note, when the treated samples were probed with anti-CR1 mAb 7G9, compared with the untreated samples there appears to be a small decrease in the signal of the main band at ~195 kDa, but a new broad and diffuse band at ~250 kDa and higher is discernible, likely representing C3dg covalently bound to CR1 (Fig. 4C). To improve on the resolution in this experiment, the proteins were also analyzed after isolation by immunoprecipitation with mAb 1H8. Indeed, as illustrated in Fig. 4D, we find that immunoprecipitation of the solubilized and hematin-treated erythrocytes with mAb 1H8 allows identification of a band ≥250 kDa that stained positive for CR1, again suggesting that some of the C3dg that deposits on the erythrocytes is covalently bound to CR1.


Figure 4
View larger version (71K):
[in this window]
[in a new window]

  		FIGURE 4. Western blots reveal that C3 fragments are covalently bound to erythrocyte membranes, and some of these fragments are directly bound to CR1. A–C, Solubilized erythrocyte membrane preparations from erythrocytes reacted with either NHS alone or with NHS plus hematin (untreated (U) and treated (T), respectively) were analyzed by Western blotting. Equal amounts of protein from each preparation were loaded on the gels. Blots were stained with either mAb 1H8 (anti-C3b/iC3b/C3dg), or with mAb 1E6 (anti-C3b/iC3b) or with anti-CR1 mAb 7G9. Positive controls were soluble iC3b or soluble CR1. In D, the erythrocytes were lysed with RIPA buffer and subjected to immunoprecipitation with mAb 1H8, followed by development with anti-CR1 mAb 7G9. The results indicate that in the treated sample, immunoprecipitation based on targeting of C3dg leads to precipitation of a higher molecular mass form of CR1, likely consisting of CR1 with C3dg covalently bound. Results displayed are for erythrocytes from the donor with high erythrocyte CR1 in Fig. 5B and are representative of similar findings for the erythrocytes of two other donors.

 
To determine whether C3 deposition could be correlated with erythrocyte CR1 levels, erythrocytes from six normal donors were subjected to three cycles of hematin/NHS treatment. After washing and dilution, the erythrocytes were probed with a mixture of biotinylated anti-CR1 mAbs (7G9 and HB8592) (34, 36), followed by PerCP-streptavidin and polyclonal FITC-goat anti-human C3 (specific for C3 and its fragments). The dot plots in Fig. 5, A and B, for one low and one high erythrocyte CR1 normal donor, reveal that within a given erythrocyte population, those erythrocytes with the most CR1 also capture the most C3 fragments, as indicated by the diagonal shape of the dot plots. In these plots, the erythrocytes were divided into eighths with respect to CR1 levels. We extended this analysis to evaluate the relationship between erythrocyte CR1 densities and C3 fragment deposition for the erythrocytes from six donors. The combined results, for all six donors, (Fig. 5C) again reveal a correlation between CR1 levels and C3 fragment deposition. Moreover, the two intersecting linear plots suggest that there may be a minimum value required for more efficient deposition of C3 fragments on the erythrocytes; CR1 densities may have to be in excess of ~1500 MESF units to capture substantial amounts of C3 fragments.

Analyses of C3 fragment deposition by fluorescence microscopy

If erythrocyte CR1 plays an important role in promoting hematin-mediated C3 fragment deposition on erythrocytes, we reasoned that the fragments would most likely deposit quite close to areas of erythrocytes containing high densities of CR1, which is organized in clusters to promote high-affinity binding to C3b-opsonized substrates (46, 48, 49). Erythrocytes were subjected to three cycles of NHS and hematin treatment, and after thorough washing, the erythrocytes were probed with a mixture of three Al488-labeled anti-CR1 mAbs and three Al594 anti-C3 fragment mAbs. Analysis by fluorescence microscopy reveals that virtually all of the deposited C3 fragments (red) are indeed located quite close to or coincident with areas of erythrocytes containing high densities of CR1 (green; Fig. 6A), but several regions displaying high densities of CR1 do not show evidence for C3 fragment deposition. To generate a positive control for colocalization, the opsonized erythrocytes were alternatively probed with a mixture composed of the same three Al488- and Al594-labeled anti-C3 fragment mAbs as were used in the first series. A higher level of colocalization is evident in these positive controls (Fig. 6B).


Figure 6
View larger version (60K):
[in this window]
[in a new window]

  		FIGURE 6. Fluorescence microscopy reveals that C3 fragments deposited on erythrocytes are located quite close to or coincident with erythrocytes CR1, but not all CR1 capture C3 fragments. Erythrocytes (40% hct in 40% NHS) were reacted with 300 µg/ml hematin (three cycles, AP) and then probed. A, Erythrocytes were reacted with a mixture of three non-cross-reacting Al488 anti-CR1 mAbs (green) and three non-cross-reacting Al594 anti-C3 fragments mAbs (red). In the merged pictures, C3 fragments are found quite close to or coincident with clustered regions of CR1, but several regions enriched in CR1 (green) do not contain deposited C3 fragments. B, The erythrocytes were reacted with a mixture containing the same three anti-C3 fragment mAbs, but in this case they were labeled with both Al488 and Al594, and all six mAb preparations were used at the same final concentrations. A higher degree of colocalization is seen. Olympus BX40 fluorescent microscope, Magnafire digital camera x100 magnification.

 
Erythrocyte rosetting with Raji cells

After multiple cycles of treatment of the erythrocytes with hematin and NHS, large amounts of C3dg are demonstrable on the erythrocytes, and therefore these opsonized erythrocytes might bind to B cells via CR2, the receptor for C3dg (28, 45). Erythrocytes were reacted for 1, 5, or 10 cycles with hematin and NHS, and after washing they were mixed with Raji cells to determine whether rosette formation would occur. Rosettes did form for the 5- and 10-cycle cells (11 and 25% rosettes, respectively) but not for the 1-cycle sample; Fig. 7 shows examples of rosettes obtained using the 10-cycle hematin-treated erythrocytes.


Figure 7
View larger version (170K):
[in this window]
[in a new window]

  		FIGURE 7. Microscopy images illustrating rosetting of Raji cells with erythrocytes (5% hct) subjected to 10 cycles of reaction with 300 µg/ml hematin in 50% NHS (AP). No rosettes were demonstrable for untreated erythrocytes or for erythrocytes reacted for only one cycle.

 
Tests with erythrocytes from other species

We performed similar tests for hematin-mediated C3 deposition with rhesus monkey serum and isolated and washed monkey erythrocytes (which express CR1) (50) and with mouse serum and isolated and washed mouse erythrocytes (which do not express CR1) (51). Findings for the monkey erythrocytes closely mirrored those we obtained for human erythrocytes, but we could detect no deposition of mouse C3 fragments on mouse erythrocytes after incubation with mouse serum and hematin (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The key observation in this study is that activation of the AP of complement by hematin promotes deposition of substantial amounts of C3 fragments on erythrocytes (Figs. 1 and 2). The deposited C3 fragments may sensitize erythrocytes for early clearance and destruction, thus providing a mechanism that may partly explain loss of uE in childhood malaria. The deposition reaction requires the intact AP of complement (Fig. 2) and our results reveal that hematin activates and consumes complement via the AP: when hematin and zymosan are mixed, C3 fragment deposition on zymosan is virtually eliminated, yet in the absence of hematin the zymosan, serves as a ready acceptor of activated C3 fragments (Tables I and II). The reaction of hematin and complement with erythrocytes in NHS is concentration dependent, with modest activities for hematin concentrations below 100 µg/ml and C3 fragment deposition reaching a maximum at ~300 µg/ml hematin (Figs. 1A and 2A). It is possible that the threshold concentration reflects the inhibitory activities of serum proteins such as hemopexin and albumin that can bind to hematin and perhaps suppress its binding to erythrocytes (2, 3, 5, 7, 9).

Omodeo-Sale et al. (21) have demonstrated that hematin, which is sufficiently hydrophobic that it binds to lipid bilayers, also binds to both intact erythrocytes and to erythrocyte ghosts. Smith and Winslow (27) have reported that several products of erythrocyte breakdown, including hematin, activate the AP of complement, and our results extend both of these previous studies. We find that hematin does indeed activate and efficiently consume complement via the AP, and our experiments indicate that the deposition of C3 fragments on erythrocytes is mediated by erythrocyte CR1. Treatment of the erythrocytes with proteases, which removes CR1, decreases C3 deposition (Fig. 3); the reaction does not occur with mouse erythrocytes that lack CR1, and we find that more C3 fragments are deposited on human erythrocytes with higher levels of CR1 (Fig. 5). It is quite likely that other proteins are also removed from erythrocytes upon protease treatment, and so the results with the proteases support a role for CR1, but other interpretations are possible. Our fluorescence microscopy experiments provide more direct evidence that CR1 plays a key role in the C3 deposition reaction because almost all deposited C3 fragments are located quite close to areas rich in CR1 (Fig. 6). The Western blots provide independent evidence that C3 fragments are indeed covalently bound to the erythrocytes (Fig. 4), and a fraction of these fragments are covalently bound to CR1. The quantitative aspects of the C3 fragment deposition reaction indicate that even after >10 cycles of treatment, C3 deposition has not reached saturation, and many more molecules of C3dg deposit on the erythrocytes than would be expected based on a 1:1 or 2:1 C3 fragment/CR1 stoichiometry (45, 52). In terms of detailed mechanisms, we cannot rule out a direct binding interaction between erythrocyte CR1 and hematin, but we are not aware of any evidence that would support this interaction. It is more likely that after either free hematin or erythrocyte-bound hematin activates complement, erythrocyte CR1 may then interact with the C3b that is generated locally during complement activation, thus allowing for direct deposition of nascent C3b at or near erythrocyte CR1.

Our findings may have important implications with respect to a possible role of complement and hematin in the pathogenesis of anemia and destruction of uE in malaria. After merozoites enter and infect erythrocytes, the destruction of the iE during schizogony, and release of by-products including hematin, followed by additional infection of other erythrocytes, occurs approximately once every 48 h (13, 53, 54). It is therefore possible that after multiple cycles of infection, the youngest erythrocytes, with the highest level of CR1 (40, 55), will most efficiently capture C3 fragments, and may be cleared most readily (12, 20, 22), due to the deposited C3dg fragments. We found that C3dg-labeled erythrocytes can form rosettes with Raji cells (Fig. 7), and it is reasonable to anticipate that these erythrocytes could be sequestered and destroyed in the spleen or perhaps in the liver, by cells which express receptors for either C3dg or possibly C3b and iC3b (56, 57, 58). It is well-established that the spleen removes parasitized erythrocytes during malarial infections (59); moreover, marginal zone B cells express high levels of CD21, the receptor for C3dg (28, 45, 58, 60), and based on our rosetting experiments (Fig. 7), the spleen may be particularly effective at removing C3dg-opsonized uE. Finally, we note that Hill et al. (61) have reported that in patients with paroxysmal nocturnal hemoglobinuria treated with the terminal complement inhibitor eculizumab, a fraction of erythrocytes in the circulation have bound C3 activation fragments, as revealed by probing the erythrocytes with a mAb specific for C3d. Their observations suggest that these erythrocytes may be cleared as a result of this opsonization, thus providing additional evidence for the mechanism that we propose.

Although we are unaware of any direct evidence that hematin levels are indeed elevated in malarial anemia, changes in several other surrogate markers have been reported in malaria and in other diseases, and these changes are consistent with release of substantial amounts of hemoglobin and hematin into the bloodstream (2, 3, 7, 9, 62). It is well-established that other severe hemolytic diseases are associated with reduced plasma concentrations of both haptoglobin and hemopexin, the plasma proteins that respectively promote the safe clearance of hemoglobin and its breakdown product heme, the most immediate and toxic products associated with erythrocytes lysis (3, 7). Moreover, as noted by Rother et al. (2), when these clearance pathways are saturated due to excessive hemolysis, hemoglobin and heme will be demonstrable in the plasma and in the urine. Several reports have documented substantial decreases in haptoglobin levels that are associated with severe malarial anemia (62, 63). In addition, Atkinson et al. (64) have found that the haptoglobin 2-2 genotype appears to be a risk factor for anemia in children with malaria compared with the 1-1 genotype, possibly due to the reduced ability of the protein to clear hemoglobin. The report of Ekvall et al. (63) provides the most direct link to the present studies. They followed children who had acute episodes of malaria for 72 h. In many of the children, plasma levels of haptoglobin and/or hemopexin were reduced, and consistent with the paradigms discussed by Rother, more than half of the children had both hemoglobinemia and hemoglobinuria. Moreover, reductions in levels of hemopexin were correlated with loss of erythrocytes. These authors reported that large amounts of complement C3 fragments, seven times higher than observed for controls, were also found on the erythrocytes of the children, which is consistent with C3 fragment deposition on erythrocytes, presumably mediated by the breakdown product hematin released from previously lysed erythrocytes. As noted, circulating hematin concentrations in malaria, as a result of schizont rupture, have not been reported, but bloodstream concentrations of ~150 µg/ml hematin (~0.23 mM) would correspond to destruction of <0.1% of circulating erythrocytes (65), indicating that the hematin concentration range in which we observe C3 fragment deposition is physiologically reasonable. We note that panhematin also promotes deposition of C3 fragments on human erythrocytes (Fig. 1E), but the usual dose of this drug (301 mg) would give concentrations in the bloodstream of <80 µg/ml, below the 100 µg/ml threshold (Fig. 1A) for high levels of deposition of C3 fragments on erythrocytes.

Experiments conducted in whole blood anticoagulated with lepirudin and mixed with hematin gave very similar results, with respect to the C3 fragment deposition reaction, to those obtained in serum (Fig. 2A), suggesting that the phenomena we have described could indeed occur in the bloodstream. If a large cohort of the youngest erythrocytes, which express the most CR1 (40, 55) are cleared, it is possible that a child with a relatively low parasite burden might suffer from sudden anemia, due to simultaneous natural clearance of old erythrocytes, along with clearance of a large fraction of the youngest, C3dg-labeled erythrocytes.

The C3 fragment deposition we have studied can be completely abrogated by the use of anti-C3b/iC3b mAb 3E7 (Fig. 2D), a mAb which we have previously demonstrated is able to block the AP of complement (32). A humanized form of mAb 3E7 has recently been described (66), and although it may not be feasible to use these mAbs in the treatment of childhood malaria, it is possible that the mAbs could be used to establish proof of principle, perhaps in recently described malaria models in nonhuman primates in which anemia is indeed demonstrable at low parasite burdens (67). We have already reported that infused mAb 3E7 can bind to C3b-opsonized B cells which remain in the circulation for brief periods after rituximab infusion in a cynomolgus monkey model (30). If proof of principle for use of mAb 3E7 to prevent erythrocyte destruction in primate models of malaria can be obtained, then it would be reasonable to determine whether less expensive small molecule inhibitors of the AP of complement might find use in the treatment of childhood malaria.


   Acknowledgment
 
We thank Barbara Schmertz for several useful suggestions and advice on key protocols.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Ellison Medical Foundation (to R.P.T.). Back

2 Address correspondence and reprint requests to Dr. Ronald P. Taylor, Department of Biochemistry and Molecular Genetics, University of Virginia, P.O. Box 800733, Charlottesville, VA 22908. E-mail address: rpt{at}virginia.edu Back

3 Abbreviations used in this paper: Pf, Plasmodium falciparum; iE, infected erythrocyte; uE, uninfected erythrocyte; NHS, normal human serum; AP, alternative pathway; Al, Alexa dye; MESF, molecules of equivalent soluble fluorochrome; CP, classical pathway; RIPA, radioimmunoprecipitation. Back

Received for publication March 19, 2007. Accepted for publication August 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Francis, S. E., D. J. Sullivan, D. E. Goldberg. 1997. Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. Annu. Rev. Microbiol. 5: 97-123. [Medline]
  2. Rother, R. P., L. Bell, P. Hillmen, M. T. Gladwin. 2005. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin. J. Am. Med. Assoc. 293: 1653-1662. [Abstract/Free Full Text]
  3. Kumar, S., U. Bandyopadhyay. 2005. Free heme toxicity and its detoxification systems in human. Toxicol. Lett. 157: 175-188. [Medline]
  4. Arruda, M. A., A. V. Graca-Souza, C. Barja-Fidalgo. 2005. Heme and innate immunity: new insights for an old molecule. Mem. Inst. Oswaldo Cruz 100: 799-803. [Medline]
  5. Hebbel, R. P., J. W. Eaton. 1989. Pathobiology of heme interaction with the erythrocyte membrane. Semin. Hematol. 26: 136-149. [Medline]
  6. Chiu, D., B. Lubin. 1989. Oxidative hemoglobin denaturation and RBC destruction: the effect of heme on red cell membranes. Semin. Hematol. 26: 128-135. [Medline]
  7. Delanghe, J. R., M. R. Langlois. 2001. Hemopexin: a review of biological aspects and the role in laboratory medicine. Clin. Chim. Acta 312: 13-23. [Medline]
  8. Goldstein, L., Z. P. Teng, E. Zeserson, M. Patel, R. F. Regan. 2003. Hemin induces an iron-dependent, oxidative injury to human neuron-like cells. J. Neurosci. Res. 73: 113-121. [Medline]
  9. Muller-Eberhard, U., J. Javid, H. H. Liem, A. Hanstein, M. Hanna. 1968. Plasma concentrations of hemopexin, haptoglobin, and heme in patients with various hemolytic diseases. Blood 32: 811-815. [Abstract/Free Full Text]
  10. Jakeman, G. N., S. Saul, W. L. Hogarth, W. E. Collins. 1999. Anaemia of acute malaria infections in non-immune patients primarily results from destruction of uninfected erythrocytes. Parasitology 119: 127-133.
  11. Menendez, C., A. F. Fleming, P. L. Alonso. 2000. Malaria-related anemia. Parasitol. Today 16: 469-476. [Medline]
  12. Stoute, J. A., A. O. Odindo, B. A. Owuor, E. K. Mibei, M. O. Opollo, J. N. Waitumbi. 2003. Loss of red blood cell-complement regulatory proteins and increased levels of circulating immune complexes are associated with severe malarial anemia. J. Infect. Dis. 187: 522-525. [Medline]
  13. Ekvall, H.. 2003. Malaria and anemia. Curr. Opin. Hematol. 10: 108-114. [Medline]
  14. Helleberg, M., B. Q. Goka, B. D. Akanmori, G. Obeng-Adjei, O. Rodriques, J. A. L. Kurtzhals. 2005. Bone marrow suppression and severe anaemia associated with persistent Plasmodium falciparum infection in African children with microscopically undetectable parasitaemia. Malaria J. 4: 56
  15. Evans, K. J., D. S. Hansen, N. van Rooijen, L. A. Buckingham, L. Schofield. 2006. Severe malaria anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes. Blood 107: 1192-1199. [Abstract/Free Full Text]
  16. Casals-Pascual, C., O. Kai, J. O. P. Cheung, S. Williams, B. Lowe, M. Nyanoti, T. N. Williams, K. Maitland, M. Molyneux, C. R. J. C. Newton, et al 2006. Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo. Blood 108: 2569-2577. [Abstract/Free Full Text]
  17. Looareesuwan, S., T. M. E. Davis, S. Pukrittayakamee, W. Supanaranond, V. Desakorn, K. Silamut, S. Krishna, S. Boonamrung, N. J. White. 1991. Erythrocyte survival in severe falciparum malaria. Acta Tropica 48: 263-270. [Medline]
  18. Price, R. N., J. A. Simpson, F. Nosten, C. Luxemburger, L. Hkirjaroen, F. T. Kuile, T. Chongsuphajaisiddhi, N. J. White. 2001. Factors contributing to anemia after uncomplicated falciparum malaria. Am. J. Trop. Med. Hyg. 65: 614-622. [Abstract]
  19. Rowe, J. A., J. M. Moulds, C. I. Newbold, L. H. Miller. 1997. P. falciparum rosetting mediated by parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388: 292-295. [Medline]
  20. Stoute, J. A.. 2005. Complement-regulatory proteins in severe malaria: too little or too much of a good thing?. Trends Parasitol. 21: 218-223. [Medline]
  21. Omodeo-Sale, F., A. Motti, A. Dondorp, N. J. White, D. Taramelli. 2005. Destabilisation and subsequent lysis of human erythrocytes induced by Plasmodium falciparum haem products. Eur. J. Haematol. 74: 324-332. [Medline]
  22. Waitumbi, J., O. Malachi, R. Muga, A. Misore, J. Stoute. 2000. Red cell surface changes and erythrophagocytosis in children with severe Plasmodium falciparum anemia. Blood 95: 1481-1486. [Abstract/Free Full Text]
  23. Abdalla, S., D. J. Weatherall. 1982. The direct antiglobin test in P. falciparum malaria. Br. J. Haematol. 51: 415-425. [Medline]
  24. Stanley, H. A., J. T. Mayes, N. R. Cooper, R. T. Reese. 1984. Complement activation by the surface of Plasmodium falciparum infected erythrocytes. Mol. Immunol. 21: 145-150. [Medline]
  25. Goka, B. Q., H. Kwarko, J. A. L. Kurtzhals, B. Gyan, E. Ofori-Adjei, S. A. Ohene, L. Hviid, B. D. Akanmori, J. Neequaye. 2001. Complement binding to erythrocytes is associated with macrophage activation and reduced haemoglobin in Plasmodium falciparum malaria. Trans. Royal Soc. Trop. Med. Hygiene 95: 545-549. [Medline]
  26. Craig, M. L., J. N. Waitumbi, R. P. Taylor. 2005. Processing of C3b-opsonized immune complexes bound to non-complement receptor 1 (CR1) sites on red cells: phagocytosis, transfer, and associations with CR1. J. Immunol. 174: 3059-3066. [Abstract/Free Full Text]
  27. Smith, D. J., R. M. Winslow. 1992. Effects of extraerythrocytic hemoglobin and its components on mononuclear cell procoagulant activity. J. Lab. Clin. Med. 119: 176-182. [Medline]
  28. Weis, J. J., T. F. Tedder, D. T. Fearon. 1984. Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes. Proc. Natl. Acad. Sci. USA 81: 881-885. [Abstract/Free Full Text]
  29. Tosic, L., W. M. Sutherland, J. Kurek, J. C. Edberg, R. P. Taylor. 1989. Preparation of monoclonal antibodies to C3b by immunization with C3bi-Sepharose. J. Immunol. Methods 120: 241-249. [Medline]
  30. Kennedy, A. D., M. D. Solga, T. A. Schuman, A. W. Chi, M. A. Lindorfer, W. M. Sutherland, P. L. Foley, R. P. Taylor. 2003. An anti-C3bi mAb enhances complement activation, C3bi deposition, and killing of CD20+ cells by rituximab. Blood 101: 1071-1079. [Abstract/Free Full Text]
  31. Kennedy, A. D., P. V. Beum, M. D. Solga, D. J. DiLillo, M. A. Lindorfer, C. E. Hess, J. J. Densmore, M. E. Williams, R. P. Taylor. 2004. Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia. J. Immunol. 172: 3280-3288. [Abstract/Free Full Text]
  32. DiLillo, D. J., A. W. Pawluczkowycz, W. Peng, A. D. Kennedy, P. V. Beum, M. A. Lindorfer, R. P. Taylor. 2006. Selective and efficient inhibition of the alternative pathway of complement by a mAb that recognizes C3b/iC3b. Mol. Immunol. 43: 1010-1019. [Medline]
  33. O’Shea, J. J., E. J. Brown, B. E. Seligmann, J. A. Metcalf, M. M. Frank, J. I. Gallin. 1985. Evidence for the distinct intracellular pools of receptors for C3b and C3bi in human neutrophils. J. Immunol. 134: 2580-2587. [Abstract]
  34. 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: varying risk groups for acquired immune deficiency syndrome. J. Clin. Invest. 78: 977-982. [Medline]
  35. Edberg, J. C., R. P. Kimberly, R. P. Taylor. 1992. Functional characterization of non-human primate erythrocyte immune adherence receptors: implications for the uptake of immune complexes by the cells of the mononuclear phagocytic system. Eur. J. Immunol. 22: 1333-1339. [Medline]
  36. 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: 190-200. [Medline]
  37. Sullivan, D. J., Jr, I. Y. Gluzman, D. G. Russell, D. E. Goldberg. 1996. On the molecular mechanism of chloroquine’s antimalarial action. Proc. Natl. Acad. Sci. USA 93: 11865-11870. [Abstract/Free Full Text]
  38. 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: 4258-4265. [Abstract]
  39. Ripoche, J., R. B. Sim. 1986. Loss of complement receptor type 1 (CR1) on ageing of erythrocytes: studies of proteolytic release of the receptor. Biochem. J. 235: 815-821. [Medline]
  40. Pascual, M., C. Danielsson, G. Steiger, J. A. Schifferli. 1994. Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS. Eur. J. Immunol. 24: 702-708. [Medline]
  41. 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: 49-59. [Medline]
  42. Gilead, Z., Y. Jeng, W. S. M. Wold, K. Sugawara, H. M. Rho, M. L. Harter, M. Green. 1976. Immunological identification of two adenovirus 2-induced early proteins possibly involved in cell transformation. Nature 264: 263-266. [Medline]
  43. Mollnes, T., O. Brekke, M. Fung, H. Fure, D. Christiansen, G. Bergseth, V. Videm, K. Lappegard, J. Kohl, J. Lambris. 2002. Essential role of the C5a receptor in E. coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation. Blood 100: 1869-1877. [Abstract/Free Full Text]
  44. 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: 20-30. [Abstract/Free Full Text]
  45. Ahearn, J. M., D. T. Fearon. 1989. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46: 183-217. [Medline]
  46. 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: 3739-3747. [Abstract]
  47. Madi, N., J. E. Steiger, J. A. Schifferli. 1991. Immune adherence and clearance of hepatitis B surface Ag/Ab complexes is abnormal in patients with systemic lupus erythematosus (SLE). Clin. Exp. Immunol. 85: 373-378. [Medline]
  48. Paccaud, J. P., J. L. Carpentier, J. A. Schifferli. 1988. Direct evidence of the clustered nature of complement receptors type 1 on the erythrocyte membrane. J. Immunol. 141: 3889-3894. [Abstract]
  49. Chevalier, J., M. D. Kazatchkine. 1989. Distribution in clusters of complement receptor type one (CR1) on human erythrocytes. J. Immunol. 142: 2031-2036. [Abstract]
  50. 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 type 1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys. J. Immunol. 155: 339-347. [Abstract]
  51. Alexander, J. J., B. K. Hack, P. N. Cunningham, R. J. Quigg. 2001. A protein with characteristics of factor H is present on rodent platelets and functions as the immune adherence receptor. J. Biol. Chem. 276: 32129-32135. [Abstract/Free Full Text]
  52. Quadri, R. A., J. A. Schifferli. 1992. Over-estimation of the number of complement receptor type 1 (CR1) on erythrocytes. Scand. J. Immunol. 36: 125-130. [Medline]
  53. Miller, L. H., D. I. Baruch, K. Marsh, O. K. Doumbo. 2002. The pathogenic basis of malaria. Nature 415: 673-679. [Medline]
  54. Okie, S.. 2005. Betting on a malaria vaccine. N. Engl. J. Med. 353: 1877[Free Full Text]
  55. Craig, M. L., M. L. Reinagel, E. N. Martin, R. Schlimgen, A. Nardin, R. P. Taylor. 1999. Infusion of bispecific monoclonal antibody complexes into monkeys provides immunologic protection against later challenge with a model pathogen. Clin. Immunol. 92: 170-180. [Medline]
  56. Helmy, K. Y., K. J. Katschke, N. N. Gorgani, N. M. Kjavin, J. M. Elliott, L. Diehl, S. J. Scales, N. Ghilardi, M. van Lookeren Campagne. 2006. CR1g: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 124: 915-927. [Medline]
  57. Yan, J., V. Vetvicka, Y. Xia, M. Hanikyrova, T. N. Mayadas, G. D. Ross. 2000. Critical role of Kupffer cell CR3 (CD11b/CD18) in the clearance of IgM-opsonized erythrocytes or soluble beta-glucan. Immunopharmacology 46: 39-54. [Medline]
  58. Whipple, E. C., R. S. Shanahan, A. H. Ditto, R. P. Taylor, M. A. Lindorfer. 2004. Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice. J. Immunol. 173: 2297-2306. [Abstract/Free Full Text]
  59. Engwerda, C. R., L. A. F. H. Beattie. 2004. The importance of the spleen in malaria. Trends Parasitol. 21: 75-80.
  60. Whipple, E. C., A. H. Ditto, R. S. Shanahan, J. R. Gatesman, S. F. Little, R. P. Taylor, M. A. Lindorfer. 2007. Low doses of antigen coupled to anti-CR2 mAbs induce rapid and enduring IgG immune responses in mice and in cynomolgus monkeys. Mol. Immunol. 44: 377-388. [Medline]
  61. Hill, A., R. P. Rother, A. M. Risitano, D. S. Cole, M. J. Cullen, S. J. Richards, C. Selleri, P. Ricci, B. Rotoli, L. Luzzatto, P. Hillmen. 2006. Blockade of intravascular hemolysis in PNH with the terminal complement inhibitor eculizumab unmasks low-level hemolysis potentially occurring through C3 opsonization. Blood 108: 291a
  62. Gyan, B., J. A. L. Kurtzhals, B. D. Akanmori, M. Ofori, B. Q. Goka, L. Hviid, C. Behr. 2002. Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children. Acta Tropica 83: 133-140. [Medline]
  63. Ekvall, H., P. Arese, F. Turrini, K. Ayi, F. Mannu, Z. Premji, A. Bjorkman. 2001. Acute haemolysis in childhood falciparum malaria. Trans. Royal Soc. Trop. Med. Hygiene. 95: 611-617. [Medline]
  64. Atkinson, S. H., K. Rockett, G. Sirugo, P. A. Bejon, A. Fulford, M. A. O’Connell, R. Bailey, D. P. Kwiatkowski, A. M. Prentice. 2006. Seasonal childhood anaemia in West Africa is associated with the haptoglobulin 2-2 genotype. PLoS Med. 3: 652-658.
  65. Carney, C. K., A. C. Schrimpe, K. Halfpenny, R. S. Harry, C. M. Miller, M. Broncel, S. L. Sewell, J. E. Schaff, R. Deol, M. D. Carter, D. W. Wright. 2006. The basis of the immunomodulatory activity of malaria pigment (hemozoin). J. Biol. Inorg. Chem. 11: 917-929. [Medline]
  66. Peng, W., X. Zhang, N. Mohammed, G. Inghirami, K. Takeshita, A. Pecora, L. L. Nardone, S. E. Pincus, L. S. Casey, G. L. Spitalny. 2005. A deimmunized chimeric anti-C3b/iC3b monoclonal antibody enhances rituximab-mediated killing in NHL and CLL cells via complement activation. Cancer Immunol. Immunother. 54: 1172-1179. [Medline]
  67. Egan, A. F., M. E. Fabucci, A. Saul, D. C. Kaslow, L. H. Miller. 2002. Aotus New World monkeys: model for studying malaria-induced anemia. Blood 99: 3863-3866. [Abstract/Free Full Text]




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 Google Scholar
Google Scholar
Right arrow Articles by Pawluczkowycz, A. W.
Right arrow Articles by Taylor, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pawluczkowycz, A. W.
Right arrow Articles by Taylor, R. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS