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RED CELLS
From the Complement Biology Group, Department of
Medical Biochemistry, University of Wales College of Medicine, Cardiff,
United Kingdom; and the Rheumatology Section, Division of Medicine,
Imperial College School of Medicine, Hammersmith Campus, London, United
Kingdom.
The glycolipid-anchored glycoprotein CD59 inhibits assembly of the
lytic membrane attack complex of complement by incorporation into the
forming complex. Absence of CD59 and other glycolipid-anchored molecules on circulating cells in the human hemolytic disorder paroxysmal nocturnal hemoglobinuria is associated with intravascular hemolysis and thrombosis. To examine the role of CD59 in protecting host tissues in health and disease, CD59-deficient
(CD59 Cell membranes express a battery of proteins that
regulate activation of the complement (C) system and provide essential
defense against damage to self.1 Regulators act either on
the enzymes of the activation pathways or on the lytic membrane attack
complex (MAC). In humans, 2 broadly expressed proteins collaborate to effect control in the activation pathways, decay-accelerating factor
(DAF; CD55) and membrane cofactor protein (MCP; CD46). A third
regulator of the activation pathways, CR1 (CD35), is expressed at
relatively few sites and acts primarily as a receptor for immune complexes. A single protein, CD59, controls MAC assembly by physical association with the nascent complex to prevent lytic pore
formation.2 In rodents, in which MCP expression is
restricted to testis,3,4 an additional broadly expressed
regulator, termed Crry, functions in the activation
pathways.5
Deficiencies of membrane C regulators in humans have provided clues to
their importance in controlling C activation in vivo. In paroxysmal
nocturnal hemoglobinuria (PNH), defects in the pathway of glycosyl
phosphatidylinositol (GPI) anchor synthesis in a hematopoietic stem
cell-derived clone cause complete absence of GPI-anchored proteins on
cells arising from the clone.6 PNH erythrocytes and other
clone-derived blood cells thus lack the GPI-anchored C regulators DAF
and CD59. Because human erythrocytes do not express the
transmembrane-anchored regulator MCP, PNH erythrocytes have no membrane
C regulators and are thus highly vulnerable to C damage. Isolated
deficiencies of DAF and CD59 have been described and provide additional
clues. Isolated deficiency of DAF has been reported in about 20 individuals in 4 kindreds.7-10 Absence of DAF was not
associated with intravascular hemolysis or any other evidence of
failure of C regulation, leading to the conclusion that absence of CD59
is the key factor in PNH. This conclusion was supported by reports of a
single individual, presenting with a PNH-like syndrome, found to have
isolated deficiency of CD59.11,12
The roles of the various regulators in tissue homeostasis have
also been assessed in rodents using blocking monoclonal antibody (mAb)
or by gene deletion. Blocking of Crry in rats caused profound hypotension and increased vascular permeability, indicating that Crry
is a key membrane regulator in this species.13
Blocking of CD59 alone caused no effect, but in combination with
blocking of Crry, it exacerbated the hypotensive and other
effects. Mice possess 2 DAF genes encoding, respectively, a
GPI-anchored form that is widely distributed and a transmembrane
form that is expressed only in testis.14 Deletion of the
gene encoding the GPI form yielded healthy mice.15 The
presence of intravascular hemolysis was not examined in this
report; however, analysis in vitro of DAF-deficient erythrocytes
revealed increased susceptibility to heterologous C attack. In stark
contrast to the apparent lack of effect of DAF deficiency, deletion of
the gene encoding Crry resulted in death in utero because of C
activation in the fetoplacental unit.16
Although these studies implicate CD59 as an important C regulator in
rodents and humans, much of the evidence is circumstantial and, for
humans, based on a single case. We therefore set out to investigate the
relevance of CD59 to C regulation by gene targeting in the mouse. We
have previously described the cloning and characterization of mouse
CD59.17 CD59 in the mouse resembles human CD59 in that it
is broadly distributed and functions to regulate MAC assembly on mouse
cells. The gene has been localized to a region of mouse chromosome 2 that is orthologous with the location of human CD59 on chromosome 11 and the gene structure is shown to resemble closely that in
humans.18 We undertook to delete the gene encoding CD59 by
standard gene-targeting technologies in mouse embryonic stem (ES) cells using a targeting vector designed to eliminate exon 3 that encodes the majority of the mature protein sequence. The derivation of CD59-deficient mice and the characterization of the
resultant phenotype are here described.
Generation of CD59-deficient mice
Collection of urine and blood
Blood was collected from mice anesthetized with halothane by tail
snipping. Blood (200 µL) was collected into EDTA (10 µL 0.5 M
stock) in graduated tubes for cell analysis and into tubes without
anticoagulant for collection of serum. When large volumes of blood were
required, mice were exsanguinated by cardiac puncture under halothane
anesthesia. Blood was either used immediately or stored in an equal
volume of Alsever solution at 4°C. Donor mouse blood was obtained by
cardiac puncture from male Balb/C mice, serum was separated immediately
after clotting on ice and stored in aliquots at Flow cytometry Blood was harvested by cardiac puncture, and its components were separated by centrifugation on Histopaque 1083 (Sigma, Poole, United Kingdom). Monoclonal antibodies to mouse CD59 and mouse DAF were developed in this laboratory and were biotinylated for use in flow-activated cell sorter (FACS) analysis.19 Phycoerythrin-conjugated streptavidin was purchased from Jackson Laboratories (West Grove, PA). All analyses were carried out on a FACScalibur, using CELLQuest software (Becton Dickinson, Oxford, United Kingdom).Immunohistochemistry Mice were killed by exsanguination under anesthesia, and tissues were quick-frozen in 2-methyl butane chilled on dry ice. Cryosections (10 µm) were cut on a Shandon cryotome (Runcorn, United Kingdom) and labeled with the biotinylated mAbs described above followed by peroxidase-conjugated extra-avidin (Sigma, Poole, United Kingdom). Cryosections were then stained as described in detail elsewhere.20Measurement of intravascular hemolysis and hemoglobinuria Mouse blood (200 µL) was collected into tubes containing 10 µL 0.5 M EDTA. The blood was promptly centrifuged, the plasma carefully removed and diluted 1:10 in 0.942 M Na2CO3. Measurement of hemolysis in the diluted samples utilized the method of Harboe.21,22 Absorbance in each of the diluted plasma samples was measured at 380 nm, 415 nm, and 450 nm in a Pharmacia tunable spectrophotometer (Amersham Pharmacia, Amersham, United Kingdom). The hemoglobin concentration (cHb in grams per liter) for each diluted sample was calculated from the following formula: cHb = 1.65 × A415 0.93 × A380 0.73 × A450. For measurement
of hemoglobinuria, freshly collected urine was aliquoted undiluted into
the wells of a 96-well plate (100 µL/well). The absorbance at 412 nm
was measured in a BioRad (Hemel Hempstead, United Kingdom) plate reader as an index of hemoglobin content.
Measurement of hematology profile and erythrocyte half-life Mouse blood (0.5 mL) was obtained by cardiac puncture and collected into EDTA-containing tubes. The blood was analyzed on an automated hematology analyser (Bayer Advia 120; Newbury, United Kingdom) that had previously been shown to generate reproducible data from mouse blood. All standard hematologic parameters, including reticulocyte count, were measured. Reticulocyte measurement on this analyser involves the measurement of light scatter and absorption by cells exposed to RNA stains, followed by automatic signal processing. The erythrocyte half-life (t1/2) in CD59 /
mice was calculated from the following formula:
t1/2CD59/ = t1/2CD59+/+ × ([% reticulocytes in
CD59+/+/% mature erythrocytes in
CD59+/+]/[% reticulocytes in
CD59//% mature erythrocytes in
CD59/]). The half-life of CD59+/+
erythrocytes was taken as 15 days, the published figure for normal erythrocytes in mice of this genetic background.23,24
Measurement of C hemolytic activity in CD59 70°C. Rabbit erythrocytes were washed in phosphate-buffered saline (PBS; Oxoid, Basingstoke, United Kingdom), and a 2% suspension was incubated with an equal volume of a 1:250 dilution in PBS of mouse anti-rabbit erythrocyte antiserum (made in house) for 15 minutes at 37°C. The
antibody-sensitized rabbit erythrocytes were washed into Veronal buffered saline (VBS++, C fixation diluent; Oxoid) at 1%
final concentration. For each mouse serum to be tested, doubling
dilutions in VBS++ were made in the wells of a 96-well
plate (50 µL/well), including zero and 100% lysis wells. The
antibody-sensitized rabbit erythrocytes (50 µL) were added to each
well, and the plate was incubated for 30 minutes at 37°C. Intact
cells were removed by centrifugation, and the absorbance at 540 nm in
the supernatant was measured as an index of hemolysis. Percentage of
hemolysis for each well and CH50 for each serum were calculated by
standard methods.25
In vitro C lysis assays on mouse erythrocytes Classical pathway assay. Washed erythrocytes were resuspended in PBS at 2% (vol/vol) and sensitized by incubation for 15 minutes on ice with a 1:100 final dilution in PBS of rabbit anti-mouse erythrocyte antiserum (generated in house). The original antiserum, made by immunization with washed normal mouse erythrocytes, contained substantial anti-CD59 reactivity as assessed by Western blotting. This reactivity was completely removed by passage of the antiserum over a column of recombinant mouse CD59 coupled to Sepharose prior to use in hemolysis assays. Sensitized erythrocytes (EAs) were washed, resuspended to 2% in VBS++, aliquoted into the wells of a 96-well plate (0.1 mL/well), and incubated at 37°C for 30 minutes with 0.1 mL dilutions of rat or mouse serum in VBS++. Zero and 100% lysis controls were included in all assays. Plates were centrifuged, and the absorbance at 412 nm in the supernatant was measured as an index of lysis. Percentage of lysis for individual wells was calculated from the following formula: % lysis = (test OD412-zero OD412 / 100% OD412-zero OD412) × 100. Cobra venom factor-mediated "reactive lysis" assay. Washed erythrocytes were resuspended in VBS++ at 1% (vol/vol), aliquoted into the wells of a 96-well plate (0.1 mL/well), and incubated with dilutions of rat or mouse serum (0.1 mL in VBS++) and cobra venom factor (CVF; 1.5 µg/mL final) for 15 minutes at 37°C. Zero and 100% lysis controls were included in all assays. Plates were centrifuged, and the absorbance at 412 nm in the supernatant was measured as an index of lysis. Percentage of lysis was calculated as described above. C5b-7 site assay. Erythrocytes were antibody sensitized as described. EAs (2% in VBS++) were then incubated with an equal volume of C8-depleted human or rat serum (diluted 1:5 in VBS++) for 15 minutes at 37°C. The C5b-7-bearing intermediates so formed (EAC5b-7) were washed and resuspended at 1% in PBS. EAC5b-7s were aliquoted into the wells of a 96-well plate (100 µL/well) and incubated for 30 minutes at 37°C with various dilutions of mouse or rat serum (100 µL in PBS). Zero and 100% lysis controls were included in all assays. Plates were centrifuged, and the absorbance at 412 nm in the supernatant was measured as an index of lysis. Percentage of lysis was calculated as described above. Acidified serum lysis assay (Ham test). The Ham test is the classical test for PNH.26 A modified Ham test, based on that described by Okada et al,11 was used in this study. Aliquots (100 µL) of fresh rat serum were placed in wells of a 96-well plate and acidified by addition of 0.2 N HCl, 10 µL/well. The pH of the serum immediately following acidification was between 6.5 and 6.8. Erythrocytes (10 µL 50% E in PBS) were added to each well, and the plate was incubated for 1 hour at 37°C. Zero and 100% lysis controls were included in all assays. Plates were centrifuged, and the absorbance at 412 nm in the supernatant was measured as an index of lysis. Percentage of lysis was calculated as described above. Administration of CVF CD59/ (3 male, 3 female) and CD59+/+
(3 male, 3 female) mice were bled by tail snipping into EDTA-containing
tubes that were kept on ice. Mice were then given CVF (5 mg/kg in PBS)
intraperitoneally. All mice were closely observed for signs of
distress. All were exsanguinated 1 hour after administration of CVF and
blood taken into EDTA as before. Plasma was separated and cHb was
measured in pre-CVF and post-CVF samples as described above.
Generation of CD59-deficient mice The mouse CD59 gene was disrupted by homologous recombination in ES cells. The targeting vector was designed to replace the major coding portion of the gene, exon 3, with the neomycin resistance gene (Figure 1). Three of 200 clones screened were successfully targeted and these were expanded and microinjected into C57BL/6 blastocysts. Chimeric mice showed germline transmission of the disrupted gene, and resultant heterozygotes were bred to give homozygotes, heterozygotes, and wild-type offspring in Mendelian ratios. Homozygous mutants were fully viable and fertile. No obvious abnormalities were seen up to 30 weeks, except that male CD59/ mice were consistently 2 to 3 g lighter in
weight than their CD59+/+ male littermates. Female mice
showed no difference in weight according to genotype.
CD59 protein is not expressed on cells or tissues from
CD59 /,
CD59+/, and CD59+/+ mice (3 in each group,
age 8 weeks at sampling) showed a 50% loss of CD59 expression in
heterozygotes and total loss of the protein in CD59/
mice (Figure 2). The expression of CD59
on leukocytes and platelets showed a similar pattern with no staining
on cells from CD59/ mice. No up-regulation of the C
regulators DAF or Crry was observed on cells from CD59/
mice (data not shown). Immunohistochemistry on a range of tissues from
4-week-old male mice, including kidney, spleen, testis, and brain,
showed total loss of CD59 expression in homozygous knockout mice
compared with CD59+/+ littermate controls (Figure
3). No obvious morphologic differences were apparent in tissues from CD59/ mice compared with
CD59+/+ mice.
Mice lacking CD59 exhibit spontaneous intravascular hemolysis and hemoglobinuria Fresh EDTA plasma samples were obtained by cardiac puncture under conditions that minimized the possibility of hemolysis ex vivo from CD59+/+, CD59+/, and CD59/
mice (6 in each group, 3 each female and male, age 10 weeks at sampling), and cHb was measured by spectrophotometry at multiple wavelengths. The mean cHb was 12.6 g/L in CD59/ mice,
6.5 g/L in CD59+/ mice, and 4.8 g/L in
CD59+/+ mice (Figure 4a). The
difference was statistically significant when comparing
CD59/ mice with either of the other groups
(P < .05 by one-way analysis of variance). Of note, cHb
was significantly higher in male CD59/ mice than in
females (14.6 g/L versus 10.6 g/L; P < .05).
Fresh urine samples were collected from CD59+/+,
CD59+/ Mice lacking CD59 are not anemic but do have an elevated reticulocyte count In a first study, blood samples from CD59/,
CD59+/, and CD59+/+ littermate mice (6 in
each group, 3 each female and male, age 10 weeks at sampling) were run
on an automated hematology analyser. Two samples were lost because of
clotting in the tube. All standard parameters were measured (Table
1). Hemoglobin concentration, red
cell number, and platelet number did not differ between the groups.
However, reticulocyte count was significantly elevated in the
CD59/ mice as compared with either of the other groups
(CD59/, 5.14%; CD59+/, 3.16%;
CD59+/+, 3.06%; P < .01). Reticulocyte
counts in CD59/ mice were lower in females than in male
mice, although this difference was not statistically significant. In a
second study, blood samples were taken from CD59/ and
CD59+/+ mice (10 in each group, all males, age 20 weeks at
sampling) and analyzed as described above. Again, with the exception of the reticulocyte count, hematologic parameters did not differ between
the groups (Table 1). In these older mice the reticulocyte count was
significantly elevated in the CD59/ mice
(CD59/, 4.58%; CD59+/+, 3.43%;
P < .001). Taking the t1/2 in wild-type mice
on this background as 15 days,24 the calculated t1/2 in CD59/ mice was 8.7 days from study
1 and 11.1 days from study 2.
Female CD59 /
(3 male, 2 female), CD59+/ (2 male, 3 female), and
CD59+/+ (2 male, 3 female) littermate mice at 10 weeks. There was no difference in hemolytic activities for same-sex
mice in the 3 groups, but male mice in each group had CH50 levels 10- to 12-fold higher than female mice. For the male mice the mean CH50
value (7 mice) was 85 hemolytic units (hu); for female mice (8 mice) the mean CH50 value was 7 hu.
Erythrocytes from mice lacking CD59 are highly sensitive to heterologous and homologous C lysis in vitro In a classical pathway assay, using an antiserum raised against mouse erythrocytes adsorbed to remove anti-CD59 reactivity, antibody-sensitized erythrocytes from CD59/ mice were
much more sensitive to C lysis using heterologous (rat) or homologous
(mouse) serum as a source of C than those from CD59+/+ mice
(Figure 5a). At serum dilutions causing
approximately 80% to 100% hemolysis of CD59/
erythrocytes, erythrocytes from CD59+/ and
CD59+/+ mice were not lysed above background by either
serum source. Of note, when the nonadsorbed antiserum was used to
sensitize, CD59+/+ erythrocytes were lysed to a much
greater extent, indicating that the anti-CD59 activity in the antiserum
blocked CD59 function (data not included). In a CVF-mediated reactive
lysis assay using heterologous (rat) or homologous (mouse) serum as
source of C, erythrocytes from CD59/ mice were again
much more sensitive to C attack than those from CD59+/+
mice (Figure 5b). In a C5b-7 site assay using mouse erythrocytes bearing human C5b-7 and either rat or mouse serum as a source of C8 and
C9, erythrocytes from CD59/ mice were also much more
sensitive to C attack than those of CD59+/+ mice (Figure
5c). Similar results were obtained using mouse erythrocytes bearing rat
C5b-7 sites (data not shown). In a modified Ham test using rat serum as
a source of C, erythrocytes from CD59/ mice were
positive in that they were lysed by acidified serum, whereas
erythrocytes from the other groups were not lysed under the same
conditions (Figure 5d). Mouse serum was not lytic in this
assay.
Administration of CVF provokes further intravascular hemolysis in
CD59 / mice at 1 hour after administration (Figure
6). The cHb also increased in female
CD59/ mice, albeit to a lesser extent. In
CD59+/+ mice, cHb was only slightly increased following CVF
treatment. None of the CVF-treated mice developed severe clinical
symptoms, although some mice in all groups showed mild signs of
shivering and piloerection.
Although it is clear that membrane C regulators provide an essential defense against damage to self cells when C is activated either through the physiologic process of "tickover" or in response to foreign particles in pathology, the relative importance of the different regulators is unclear. Attempts have been made to elucidate the relative contributions of the various regulators to homeostasis in vivo by neutralizing single or multiple regulators in rodents using specific antibodies.13,27,28 These studies have demonstrated a key role for Crry in the regulation of C activation and the importance of CD59 as a second-line defense when activation is inadequately regulated in the activation pathways. Although neutralization of CD59 alone in vivo in rats caused only minor disturbance, in combination with neutralization of Crry, an important role for CD59 was revealed. Indeed, neutralization of CD59 alone in a normal rat knee was sufficient to trigger an acute arthritis, presumably because of the high level of tickover activation occurring at this site.29 Further, neutralization of CD59 in the rat kidney caused a marked exacerbation of disease in a model of glomerulonephritis.30 Indications of the relative importance of membrane regulators in man
have been gleaned from studies of the acquired hemolytic disorder PNH
and rare inherited deficiencies. In PNH, a mutation in a hematopoietic
stem cell gives rise to an expanding clone of blood cells that lack
glycolipid-anchored molecules.6,31 Erythrocytes and other
blood cells derived from the clone thus lack DAF and CD59.
Human erythrocytes also do not express MCP, making PNH erythrocytes
particularly susceptible to C damage because they lack all intrinsic C
regulators. Individuals present with hemoglobinuria, anemia, and,
sometimes, thrombotic problems because of involvement of platelets and
neutrophils. Isolated deficiency of DAF has been described in 4 families and is not associated with loss of C regulation, implying that
lack of DAF is not itself responsible for the hemolytic and thrombotic
features of PNH.7-9 In contrast, isolated CD59 deficiency
has been reported in a single individual, a 22-year-old Japanese man
who had presented with a classical PNH-like syndrome at the unusually
young age of 13 and continued to experience episodes of hemolysis and
intravascular thrombosis.12,32 This single case implicated
lack of CD59 as the key factor in causing pathology in PNH. However,
given that only a single case of CD59 deficiency has been described and
relatively few studies of the susceptibility of CD59 In the present study, we undertook to delete the gene encoding CD59 in
mice by homologous recombination in ES cells. Mouse CD59 had previously
been identified, characterized, and cloned in this
laboratory,17 and the genomic structure
ascertained,18 enabling design of a gene-targeting
cassette. Heterozygote crosses produced homozygote
CD59 We have used the CD59 In marked contrast to the findings in humans with PNH and the
CD59-deficient individual, CD59 Male CD59 It must be stressed that the phenotype observed in the
CD59 The phenotypes in both the CD59 A recent report describes the presence in mice of a second gene,
designated cd59b, encoding a CD59-like protein expressed only in testis.39 We do not at present know whether this
gene is expressed in the CD59 CD59 is the only membrane regulator of the membrane attack pathway to
be characterized. Lack of CD59 will thus render all cells exposed to
plasma susceptible to damage by MAC. The spontaneous intravascular
hemolysis observed in the CD59
Submitted October 11, 2000; accepted March 13, 2001.
Supported by the Wellcome Trust through the award of a Senior Clinical Fellowship (grant no. 016668 to B.P.M.) and Programme support (grant no. 054838 to M.J.W. and M.B.).
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: B. Paul Morgan, Complement Biology Group, Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom; e-mail: morganbp{at}cardiff.ac.uk.
1. Morgan BP, Harris CL. Complement regulatory proteins. London United Kingdom: Academic Press; 1999. |
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Abstract
Introduction
