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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-06-1875.
IMMUNOBIOLOGY
From the Center for Experimental Therapeutics,
Department of Pharmacology, Department of Pathology and Laboratory
Medicine, and Department of Medicine, University of Pennsylvania School
of Medicine, Philadelphia; and the Department of Medicine,
Washington University School of Medicine, St Louis, MO.
The role of complement in the pathogenesis of autoimmune hemolytic
anemia (AIHA) has been controversial and may depend on a number of
factors, including the affinity and isotype of the pathogenic
antibodies involved. We have recently shown that mouse erythrocytes
deficient in the membrane C3 regulatory protein, complement receptor
1-related gene/protein y (Crry), but not decay-accelerating factor (DAF), were spontaneously eliminated in vivo by
complement. Here, by generating a mouse deficient in both DAF and Crry,
we further delineated the roles of Crry and DAF in regulating
alternative and classical pathway C3 activation. By using
immunoglobulin-, Fc In autoimmune hemolytic anemia (AIHA), binding of
autoantibodies to erythrocyte surface antigens results in abnormally
accelerated clearance of erythrocytes through either extravascular or
intravascular hemolysis.1 Although both the Fc receptor
(FcR) pathway and the complement system are thought to be involved in
the hemolytic process,1-3 several recent studies in the
mouse have indicated a primary role for the FcR pathway and questioned
the relevance of complement in this process.4-7 Thus,
injection of rabbit polyclonal antimouse erythrocyte immunoglobulin G
(IgG) or a murine IgG2a antierythrocyte autoantibody
caused hemolytic anemia in wild-type and C3 knockout mice but not in
FcR We have recently shown that mouse erythrocytes deficient in one of the
membrane-bound complement-regulatory proteins, complement receptor
1-related gene/protein y (Crry), were spontaneously eliminated in vivo
by the complement system.9 This finding suggested that membrane complement-regulatory proteins play a critical role in determining erythrocyte sensitivity to complement damage under normal
conditions, and probably in AIHA. Crry is a rodent (mouse and
rat)-specific membrane complement regulator that can inhibit both
antibody-induced classical pathway and alternative pathway complement
activation.10,11 It overlaps in function with
decay-accelerating factor (DAF) and membrane cofactor
protein,12 2 membrane C3 inhibitors that are universally
expressed in mammalian species including the mouse and human. Although
the expression of membrane cofactor protein in the mouse is restricted
to the testis,13,14 both DAF and Crry are expressed on
mouse erythrocytes.9,10,15 The observation that
Crry-deficient, but not DAF-deficient, mouse erythrocytes were
spontaneously cleared by complement9 raised the question
of whether DAF is redundant on mouse erythrocytes. Here, by
creating a mutant mouse that is deficient in both DAF and Crry, we have
delineated the relative roles of Crry and DAF on mouse erythrocytes in
regulating alternative and antibody-induced complement activation. By
using a number of mutant mouse lines as transfusion recipients, we also
addressed the mechanism by which Crry/DAF-deficient erythrocytes are
spontaneously cleared from the circulation. We describe here that
although Crry played a more critical role than DAF in regulating the
alternative pathway of complement, DAF and Crry were equally active in
preventing antibody-induced runaway complement activation on mouse
erythrocytes. We also determined that Crry/DAF-deficient mouse
erythrocytes were cleared spontaneously by the alternative pathway of
complement, via complement receptor-mediated extravascular hemolysis
without involving the terminal lytic complex (C5b-9). Finally, we
demonstrated that Crry/DAF-deficient erythrocytes opsonized with a
murine antierythrocyte IgG2a autoantibody were eliminated more rapidly
by the complement system than by the FcR pathway. These findings shed
new light on the relative roles of Crry and DAF in vivo, and suggested
that membrane complement regulators prevent extravascular hemolysis under normal physiologic conditions and influence the hemolytic pathway
in AIHA.
Mice
FACS analysis
C3 deposition assays Antibody-induced C3 deposition on mouse erythrocytes was performed as previously described.9 Erythrocytes (1 × 106 cells in 200 µL PBS) were opsonized with a monoclonal mouse IgG2a erythrocyte autoantibody 34-3C (50 µg/mL). The hybridoma for 34-3C was originally derived from the autoimmune NZB mice8,22 and was kindly provided by Dr Raphael Clynes (The Rockefeller University, New York, NY). The 34-3C antibody was purified from ammonium sulfate-precipitated concentrated tissue culture supernatant followed by protein A-G affinity chromatography. Antibody-opsonized cells were incubated with mouse complement (prepared in-house from 129J/C57Bl/6 mice, used at 1:40 dilution) in gelatin-veronal buffered saline (GVBS++) (Sigma) at 37°C for 30 minutes. Cells were washed 3 times in PBS and then stained with a FITC-conjugated goat antimouse C3 (ICN, Aurora, OH) and analyzed by FACS for C3 deposition.Assessment of erythrocyte survival in vivo To determine the clearance of complement regulator-deficient erythrocytes in vivo, cells (from 150 µL blood) from Crry/C3 /, DAF/C3/, or
DAF/Crry/C3/ mice were labeled ex vivo with biotin as
previously described9 and were introduced into
various host mice via the tail vein. In some experiments, the
biotinylated cells were also opsonized with monoclonal antibody
(mAb) 34-3C, an antimouse erythrocyte IgG2a autoantibody
(1 × 107 cells in 200 µL containing 50 µg/mL 34-3C).
Blood samples were collected at 5 minutes after erythrocyte infusion
and at various indicated time points thereafter. Collected
erythrocytes were stained with R-(PE)-conjugated
streptavidin (Molecular Probes, Eugene, OR), and the
percentage of biotinylated cells was determined at each time point.
To study erythrophagocytosis, DAF/Crry/C3
Relative activities of Crry and DAF in regulating spontaneous and antibody-induced complement activation on mouse erythrocytes In a previous study, we observed that when transfused into wild-type (C3-sufficient) mice, Crry/C3/ but not
DAF/C3/ mouse erythrocytes were spontaneously
eliminated by complement.9 Experiments using male and
female C57Bl/6, 129J, or mixed 129J/C57Bl/6 mice as recipients showed
that this phenomenon was not strain- or sex-dependent (data not
shown). Because both Crry and DAF function as regulators of C3
activation of the classical and the alternative pathways,12,23,24 the finding that
Crry/C3/ but not DAF/C3/ erythrocytes
were susceptible to complement clearance was rather unexpected and
implied differential activities of Crry and DAF on mouse erythrocytes.
To further delineate the relative roles of Crry and DAF on mouse
erythrocytes, we generated a Crry/DAF/C3/ mouse by
crossbreeding Crry/C3/ and DAF/C3/
mice. The successful generation of Crry/DAF/C3/ mice
was indicated by the complete absence of Crry and DAF on their
erythrocytes, as shown by FACS analysis (Figure
1), and confirmed by Southern blot
analysis of tail DNA (data not shown). We then carried out transfusion
experiments to compare the sensitivities of
Crry/DAF/C3/, Crry/C3/, and
DAF/C3/ mouse erythrocytes to complement damage in
vivo. Figure 2A demonstrates that
Crry/DAF/C3/ cells appeared to be more vulnerable than
Crry/C3/ cells to spontaneous elimination in
vivo, as lower numbers of Crry/DAF/C3/ cells were
consistently recovered at 5 minutes after transfusion into
wild-type (C3-sufficient) mice. Nevertheless, the overall clearance
kinetics of Crry/DAF/C3/ cells, when followed between 5 minutes and 3 days, was very similar to that of
Crry/C3/ cells (Figure 2B). This experiment confirmed
that Crry plays a more critical role than DAF in preventing spontaneous
complement damage of mouse erythrocytes.9 It also
suggested, however, that although DAF deficiency alone was
inconsequential (Figure 2A),9,15 the absence of DAF in the
context of Crry deficiency may have a detectable, although by no means
dramatic, influence on the susceptibility of these cells to spontaneous
complement attack.
We next evaluated the relative activities of Crry and DAF in regulating
antibody-induced (classical pathway) complement activation on mouse
erythrocytes. For this assay, C3 Crry/DAF/C3 /
erythrocytes,9 the elimination of transfused
Crry/DAF/C3/ erythrocytes in wild-type recipient mice
was C3-dependent (Figure 2D). To determine if this process was related
to the phenomenon of natural antibody-mediated transfusion reaction,
which involved the activation of the classical pathway of complement,
we first transfused Crry/DAF/C3/ erythrocytes into
Ig/ mice. These mice lacked the production of IgM and
class-switched antibodies19 and should not be able to
mount a classical pathway of complement-mediated transfusion reaction.
Figure 3 shows that Crry/DAF/C3/ erythrocytes were cleared normally in
Ig/ mice, as they were in wild-type recipient mice.
Thus, elimination of Crry/DAF/C3/ erythrocytes was
independent of the antibody repertoire of the recipient. To exclude the
possibility that Crry/DAF/C3/ erythrocytes might have
autoantibodies that were prebound to their surface and that could
have activated the classical pathway of complement upon
transfer into a C3-sufficient host, we studied the survival of
Crry/DAF/C3/ cells in C4/ mice. These
mice had targeted deletion of the C4 gene and thus lacked the classical
pathway of complement.18 Figure 3A shows that C4
deficiency similarly did not protect Crry/DAF/C3/ cells
from spontaneous elimination. This result suggested that elimination of
Crry/DAF/C3/ cells was mediated by the alternative
rather than the classical pathway of complement. Finally, transfusion
of Crry/DAF/C3/ erythrocytes into
FcR / mice demonstrated normal kinetics of clearance
(Figure 3A), confirming that the FcR pathway was also not involved in
the spontaneous destruction of Crry/DAF/C3/
cells.
Crry/DAF/C3 / erythrocytes
were destroyed via intravascular hemolysis by the lytic pathway of
complement (C5b-9) or via extravascular hemolysis through complement
receptor (CR)-mediated erythrophagocytosis. Figure 3B shows
that when Crry/DAF/C3/ erythrocytes were transfused
into C5/ mice, they were eliminated as usual, with
kinetics not dissimilar to that observed in wild-type recipient mice.
Thus, an intact lytic pathway was not required, implying that
Crry/DAF/C3/ erythrocytes were cleared primarily, if
not exclusively, through CR-mediated extravascular hemolysis. To
confirm this implication, Crry/DAF/C3/ cells
were labeled with CFSE, and their possible sequestration in the liver
and spleen of wild-type recipient mice was examined. The use of CFSE
allowed the direct examination of frozen liver and spleen sections
under a fluorescence microscope, and pilot experiments had established
that CFSE-labeled Crry/DAF/C3/ cells had clearance
kinetics similar to that of biotin-labeled cells when
transfused into wild-type mice (data not shown). Figure 3C,E shows
that, 24 hours after transfusion, CFSE+ cell clusters
were observed in the spleens of wild-type recipient mice. No trapping
of CFSE-labeled cells was observed in the spleens of
C3/ recipient mice (Figure 3D,F), which failed to clear
Crry/DAF/C3/ cells from their circulation (Figure 2B).
No fluorescence above background level was observed in the liver of
either wild-type or C3/ recipient mice (data not
shown), suggesting that complement-opsonized Crry/DAF/C3/ erythrocytes were removed by splenic
rather than liver phagocytes.
Membrane regulators of complement can significantly influence the hemolytic pathway and kinetics in AIHA The marked sensitivity displayed by Crry/DAF/C3/
erythrocytes to antibody-induced complement activation in vitro (Figure
2C) suggested that membrane regulators of complement may significantly influence the hemolytic pathways in AIHA, that is, the relative contribution of the FcR versus the complement pathway in the
pathogenesis of AIHA. To test this hypothesis, we compared the relative
efficiency of the FcR pathway with that of the complement
pathway in clearing antibody-opsonized Crry/DAF/C3/
erythrocytes in vivo. To isolate the function of the FcR from the
complement pathway and vice versa, we used C3/ or
FcR/ mice, respectively, as recipients for
transfusing antibody-opsonized Crry/DAF/C3/
erythrocytes. Figure 4 shows that when
Crry/DAF/C3/ erythrocytes opsonized in vitro with 50 µg/mL 34-3G (IgG2a autoantibody) were transfused into
FcR/ mice (complement-dependent clearance), they
were rapidly eliminated within the first 5 minutes. In contrast, when
such cells were transfused into C3/ mice (FcR-dependent
clearance), the kinetics of clearance was much slower. A high
percentage of the transfused cells remained at 5 minutes, and little
change was detected between 5 minutes and 3 hours (Figure 4). Thus, in
the absence of Crry and DAF from the cell surface, complement was shown
to be a more efficient pathway for disposing of erythrocytes
opsonized with a complement-fixing autoantibody.
Murine erythrocytes express 2 functionally overlapping membrane regulators of C3 activation, DAF and Crry, as well as a membrane inhibitor of the terminal complement attack complex, CD59. In a previous study, we found that targeted deletion of the DAF and CD59 genes in the mouse significantly increased the sensitivity of its erythrocytes to antibody-induced complement lysis but, surprisingly, had minimal effect on its erythrocyte sensitivity to spontaneous complement attack.9 In contrast, deficiency of Crry on the mouse erythrocytes rendered them susceptible to spontaneous elimination by a C3-dependent mechanism.9 These findings questioned the relative roles of Crry and DAF on murine erythrocytes and made us wonder about the mechanism by which Crry-deficient mouse erythrocytes were spontaneously cleared from the circulation. In the present study, we have addressed these questions, as well as the
concept that membrane regulators of complement may also significantly
influence the hemolytic pathways in AIHA. To define the relative roles
of Crry and DAF, we generated a Crry/DAF/C3 Using a number of knockout mouse strains as recipients in the
transfusion experiments, we also defined the pathways by which Crry/DAF-deficient mouse erythrocytes were spontaneously cleared from
the circulation. We demonstrated that the clearance of such cells was
dependent on C3 but not on C4 or C5, suggesting that the classical
pathway or the terminal lytic cascade of complement was not involved.
That the genetic background of the recipient mice (129J, C57Bl/6) had
no effect on the fate of transfused Crry/DAF/C3 Our study has demonstrated that membrane complement regulators are
absolutely required for protecting erythrocytes from spontaneous complement damage. Whether these proteins are also required for homeostatic protection of other types of adult mouse tissues remains to
be examined although, as alluded to earlier, Crry-deficient mouse
embryos, like erythrocytes, were also susceptible to spontaneous complement attack.16 The revelation of an essential role
of Crry and DAF in protecting erythrocytes under normal physiologic conditions raised the issue of relevance of membrane complement regulators in autoimmune hemolytic anemia. Although complement has long
been considered to be a major effector pathway in the pathogenesis of
AIHA,1-3 its importance in AIHA, relative to that of the
FcR pathway, has been questioned by a number of recent studies in a
murine AIHA model.5-7 It now appears that the affinity and
IgG isotype of the pathogenic antibodies may determine, to a large
extent, the relative contribution of the 2 pathways.8 Thus, the murine mAb 34-3C, an IgG2a antierythrocyte autoantibody, caused a predominantly FcR-mediated hemolysis in which the complement system played a secondary role.8 In comparison, an
IgG1-class switch variant of the same antibody invoked only the Fc
We thank Dr Noriko Okada for antimouse DAF antibodies, Dr Michael
Holers for the antimouse Crry antibody, Dr Rick Wetsel for C3 knockout
breeder mice, and Dr Alan Schreiber for Fc
Submitted June 26, 2002; accepted July 17, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-06-1875.
Supported by an Established Investigator Award from the American Heart Association and by National Institutes of Health grants AI 44970, AI 49344 (W.C.S.), and AI 40576 and AI 44912 (H.M.).
H.M. and T.M. contributed equally.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wen-Chao Song, Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 1351 BRBII/III, 421 Curie Blvd, Philadelphia, PA 19104; e-mail: song{at}spirit.gcrc.upenn.edu.
1.
Frank MM.
Pathophysiology of immune hemolytic anemia.
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1977;87:210-222 2. Schreiber AD, Frank MM. Role of antibody and complement in the immune clearance and destruction of erythrocytes, II: molecular nature of IgG and IgM complement-fixing sites and effects of their interaction with serum. J Clin Invest. 1972;51:583-589[Medline] [Order article via Infotrieve]. 3. Schreiber AD, Frank MM. Role of antibody and complement in the immune clearance and destruction of erythrocytes, I: in vivo effects of IgG and IgM complement-fixing sites. J Clin Invest. 1972;51:575-582[Medline] [Order article via Infotrieve].
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Shibata T, Berney T, Reininger L, et al.
Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause autoimmune hemolytic anemia by two distinct pathogenic mechanisms.
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Sylvestre D, Clynes R, Ma M, Warren H, Carroll MC, Ravetch JV.
Immunoglobulin G-mediated inflammatory responses develop normally in complement-deficient mice.
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Meyer D, Schiller C, Westermann J, et al.
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Azeredo da Silveira S, Kikuchi S, Fossati-Jimack L, et al.
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Miwa T, Zhou L, Hilliard B, Molina H, Song WC.
Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack.
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2002;99:3707-3716 10. Li B, Sallee C, Dehoff M, Foley S, Molina H, Holers VM. Mouse Crry/p65: characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF. J Immunol. 1993;151:4295-4305[Abstract]. 11. Holers VM, Kinoshita T, Molina H. The evolution of mouse and human complement C3-binding proteins: divergence of form but conservation of function. Immunol Today. 1992;13:231-236[CrossRef][Medline] [Order article via Infotrieve].
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Kim YU, Kinoshita T, Molina H, et al.
Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein.
J Exp Med.
1995;181:151-159 13. Miwa T, Nonaka M, Okada N, Wakana S, Shiroishi T, Okada H. Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1. Immunogenetics. 1998;48:363-371[CrossRef][Medline] [Order article via Infotrieve]. 14. Tsujimura A, Shida K, Kitamura M, et al. Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells. Biochem J. 1998;330:163-168.
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Sun X, Funk CD, Deng C, Sahu A, Lambris JD, Song WC.
Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.
Proc Natl Acad Sci U S A.
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Xu C, Mao D, Holers VM, Palanca B, Cheng AM, Molina H.
A critical role for murine complement regulator crry in fetomaternal tolerance.
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2000;287:498-501 17. Circolo A, Garnier G, Fukuda W, et al. Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA. Immunopharmacology. 1999;42:135-149[CrossRef][Medline] [Order article via Infotrieve]. 18. Fischer MB, Ma M, Goerg S, et al. Regulation of the B cell response to T-dependent antigens by classical pathway complement. J Immunol. 1996;157:549-556[Abstract]. 19. Kitamura D, Roes J, Kuhn R, Rajewsky K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 1991;350:423-426[CrossRef][Medline] [Order article via Infotrieve]. 20. Takai T, Li M, Sylvestre D, Clynes R, Ravetch JV. FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell. 1994;76:519-529[CrossRef][Medline] [Order article via Infotrieve]. 21. Ohta R, Imai M, Fukuoka Y, Miwa T, Okada N, Okada H. Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes. Microbiol Immunol. 1999;43:1045-1056[Medline] [Order article via Infotrieve]
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Fossati-Jimack L, Reininger L, Chicheportiche Y, et al.
High pathogenic potential of low-affinity autoantibodies in experimental autoimmune hemolytic anemia.
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© 2002 by The American Society of Hematology.
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  T. Miwa, L. Zhou, Y. Kimura, D. Kim, A. Bhandoola, and W.-C. Song Complement-dependent T-cell lymphopenia caused by thymocyte deletion of the membrane complement regulator Crry Blood, March 19, 2009; 113(12): 2684 - 2694. [Abstract] [Full Text] [PDF]
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D. D. Kim, T. Miwa, Y. Kimura, R. A. Schwendener, M. van Lookeren Campagne, and W.-C. Song Deficiency of decay-accelerating factor and complement receptor 1-related gene/protein y on murine platelets leads to complement-dependent clearance by the macrophage phagocytic receptor CRIg Blood, August 15, 2008; 112(4): 1109 - 1119. [Abstract] [Full Text] [PDF]
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Y. Kimura, T. Miwa, L. Zhou, and W.-C. Song Activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement Blood, January 15, 2008; 111(2): 732 - 740. [Abstract] [Full Text] [PDF]
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 D. D. Kim, T. Miwa, and W.-C. Song Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack J. Immunol., October 15, 2006; 177(8): 5558 - 5566. [Abstract] [Full Text] [PDF]
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D. Spitzer, J. Unsinger, D. Mao, X. Wu, H. Molina, and J. P. Atkinson In Vivo Correction of Complement Regulatory Protein Deficiency with an Inhibitor Targeting the Red Blood Cell Membrane J. Immunol., December 1, 2005; 175(11): 7763 - 7770. [Abstract] [Full Text] [PDF]
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 J. Liu, T. Miwa, B. Hilliard, Y. Chen, J. D. Lambris, A. D. Wells, and W.-C. Song The complement inhibitory protein DAF (CD55) suppresses T cell immunity in vivo J. Exp. Med., February 14, 2005; (2005) jem.20040863. [Abstract] [Full Text] [PDF]
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M. Jasinski, P. Pantazopoulos, R. P. Rother, N. van Rooijen, W.-C. Song, H. Molina, and M. Bessler A novel mechanism of complement-independent clearance of red cells deficient in glycosyl phosphatidylinositol-linked proteins Blood, April 1, 2004; 103(7): 2827 - 2834. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
; n = 4),
Crry/C3
; n = 4), and
Crry/DAF/C3
; n = 4) erythrocytes recovered from
age-matched wild-type (C57Bl/6) recipient mice. Erythrocytes were
pooled from 6 to 8 donor mice and labeled with biotin, and
3 × 108 cells were transfused into each recipient mouse.
(B) Transformation of data from panel A to show the clearance kinetics
from 5 minutes (taken as 100%) to 3 days after transfusion. (C)
Sensitivity of C3
) with 50 µg/mL mAb 34-3C, and transfused into
Fc