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Departments of
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Medicine,
Microbiology and Immunology,
Physiology, and
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Microbiology, Morehouse School of Medicine, Atlanta, GA 30310
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pure lipids generally are poorly immunogenic, and immunization of
animals with pure phospholipid (PL) in adjuvant does not induce aPL
production (6, 7). Some investigators, however, produced
aPL experimentally in animals, using complex methods such as frequent
i.v. injections of cardiolipin (CL) coupled to methylated BSA
(8), i.p. immunization with CL coupled to monoclonal
aPL-coated Staphylococcus aureus (9), and
intrasplenic immunization with Salmonella minnesota coated
with PL (10). APL Abs have also been induced in mice by
immunization with vesicles containing various lipids and lipid A
(11). The aPL in all these experiments were not shown to
be pathogenic, and not all the above mentioned methods were reproduced
by other investigators. In 1992, we described for the first time a
simple method for the induction of very high levels of aPL in
laboratory animals by immunization with heterologous,
ß2-glycoprotein I
(ß2GPI) (12). In 1990, three
groups independently reported that a plasma protein is a cofactor for
aPL binding to cardiolipin (13, 14, 15). This protein was
identified as ß2GPI, also known as
apolipoprotein H. ß2GPI is a heavily
glycosylated single-chain normal plasma protein of 326 aa with a
molecular mass of 
50 kDa. It is a non-complement-binding member of
the complement control protein repeat superfamily that has in common
four short consensus repeats (SCR) of 60 aa each, also known as
Sushi domains. Each domain is formed by four disulfide-bonded cysteines
in a pattern of Cys 1–3 and Cys 2–4. ß2GPI
also has a fifth SCR that forms a modified Sushi domain and contains a
PL-binding site. ß2GPI is believed to be part
of the epitope to which aPL Abs bind. Mice and rabbits immunized with
ß2GPI produced high levels of aPL Abs in
addition to Abs against ß2GPI
(12). Binding of these aPL Abs to PL was enhanced by
addition of ß2GPI similar to the autoimmune
human aPL Abs (16). Our findings were reproduced by other
investigators (17, 18) and
ß2GPI-induced aPL Abs were shown to be
pathogenic in certain strains of mice (19, 20, 21, 22).
We demonstrated that immunization of mice with purified human or bovine ß2GPI, but not murine ß2GPI, induced aPL production (12). We hypothesized that in vivo binding of foreign ß2GPI to self-PL formed immunogenic complexes against which aPL were produced. To better understand the mechanisms involved in aPL production after immunization with ß2GPI, we tried to determine which part of ß2GPI is responsible for the induction of pathogenic aPL Abs. In the present study, we attempted to induce aPL in mice with synthesized PL-binding proteins constructed by coupling synthetic peptides representing the PL-binding region of the ß2GPI to carrier proteins such as BSA and also study whether these aPL Abs are pathogenic.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 15-aa peptide that spans
Gly274–Cys288 in the fifth domain of human
ß2GPI, and which we called GDKV, was obtained
(Q.C. Biochemical, Hopkins, MA). This part of
ß2GPI was previously shown to bind CL and other
anionic PL (23). This peptide contains four lysines
flanked by hydrophobic residues (shown as double-underlined) (Table I
). A modified version of GDKV in which
all the residues between Cys281 and Cys288 were
replaced with lysine was also prepared. This peptide binds PL more
strongly than GDKV (24). This peptide was called
GDKV2 (Table I). Conjugates of these peptides to
BSA and keyhole limpet hemocyanin (KLH) were also obtained. Binding of
these peptides to PL was determined by measuring their competition with
ß2GPI in binding to PL-coated plates.
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Binding of these synthetic peptides to PL was confirmed by competitive PL-binding ELISA. Microtiter plates were coated with 50 µl/well CL in ethanol, 50 µg/ml, overnight at 4°C. Plates were washed three times with PBS (150 µl/well), and blocked for 2 h with 2% OVA in PBS (100 µl/well). Then, duplicates of 50-µl aliquots of serial dilutions of human ß2GPI in 2% OVA/PBS (12.5–800 nM final concentration) were added to each well, followed by equal volumes of PBS alone (control) or PBS containing peptide, 6 µM (final concentration). Plates were incubated for 3 h at 4°C, followed by three washes with PBS. Binding of ß2GPI to CL was detected with a monoclonal anti-ß2GPI Ab, produced in our laboratory (22), which did not react with GDKV, diluted 1:100 in 2% OVA/PBS, followed by alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO), 1:1000 (one h incubation each), and the color reaction was obtained by adding the specific substrate (p-nitrophenyl phosphate). The degree of competition with ß2GPI and thus the binding of the peptide to CL were determined by comparing the optical densities of wells containing ß2GPI alone (controls) with those of wells containing ß2GPI plus peptides. The percentage inhibition was calculated as: % inhibition = [(ODcontrol - ODtest)/ODcontrol] x 100.
Direct binding of GDKV peptides to phospholipid
After the immunization experiments, when mouse antisera to GDKV peptides were available, binding of these peptides to CL-coated plates was directly confirmed by ELISA. Plates were coated with CL blocked with 2% OVA/PBS and incubated with GDKV peptides in PBS at 20 µg/ml or PBS alone. Mouse antisera to GDKV and GDKV2 were diluted 1:100 in 1% OVA/PBS, added to the corresponding wells, and incubated for 1 h, followed by alkaline phosphatase-conjugated anti-mouse IgG and then p-nitrophenyl phosphate as described above.
Immunization with synthetic peptides
Groups of 12 female NIH/Swiss mice were immunized with the following Ags in Freund’s adjuvant: human ß2GPI, 30 µg/mouse (positive control); free GDKV, GDKV conjugated to KLH, GDKV conjugated to BSA; GDKV2-BSA; KLH; or BSA alone.
Dose determination of immunogens
Induction of aPL after immunization with ß2GPI appears to be a function of the ability of this protein to bind acidic PL. Thus, the dose of GDKV and GDKV2 was based in part on their m.w. as well as their PL-binding ability. After two booster injections with 2- weekly intervals, mice were bled, and sera were tested by ELISA for Ab activity.
Determination of aPL, anti-ß2GPI, and anti-peptide Abs
Anticardiolipin activity was determined in the sera of the mice
and supernatant of the hybridoma by a standard anticardiolipin ELISA,
using alkaline phosphatase-conjugated anti-mouse IgG, as described
elsewhere (12). Supernatant was tested at 1:5 and 1:10
dilutions, and the mouse sera were tested at a 1:50 dilution in 10%
adult bovine serum (10% ABS-PBS). The color reaction was stopped when
a positive control of 100 IgG antiphospholipid units reached an OD
of 1.0. Similarly, the presence of Abs to
ß2GPI, peptides, or BSA was determined with the
use of plates coated with these Ags, blocked with 2% OVA/PBS. This
buffer was also used as a diluent for the mouse sera when tested for
anti-BSA activity. A rabbit antiserum to human
ß2GPI, produced in our laboratory
(12), was used as a positive control for
anti-ß2GPI Abs.
Monoclonal Abs
To study the pathogenic effects of GDKV-induced aPL Abs, mAbs were developed. Spleen cells from a GDKV-BSA-immunized aPL-producing mouse were fused with P3X 63Ag 8.653 (American Type Culture Collection, Manassas, VA CRL 1580), a nonsecreting myeloma cell line, using polyethylene glycol (25). Ab-secreting hybridoma clones were rendered monoclonal by limiting dilutions method, and the mAbs were tested for aPL and anti-ß2GPI activity by ELISA. One GDKV-induced mAb with aPL and anti-ß2 GPI activity was used in the in vivo thrombus enhancement and endothelial cell activation studies.
Purification of mouse mAb from the hybridoma supernatant
Mouse mAb was purified from the supernatant of the hybridoma by affinity chromatography, utilizing an anti-mouse IgG or IgM affinity column. The purified mAbs were dialyzed against Tris-buffered saline, pH 7.4, and the concentration of the protein was determined by the Lowry method (Sigma). Samples were filtered sterile before injection into the mice.
In vivo experiments
The effects of aPL Abs on thrombosis was studied by examining the dynamics of thrombus formation in the exposed femoral vein (26, 27, 28) and their ability to activate endothelial cells in vivo was determined by examining white blood cell (WBC) adhesion to endothelium in an exposed cremaster muscle (29, 30). Both experiments were performed in the same treated mouse (30).
Animals and injection protocol. Normal male CD-1 (outbred) mice weighing 30–40 g (Charles River Laboratories, Wilmington, MA) were used for these studies. The animals were housed in the Animal Care (American Association of Laboratory Animal Care-approved) facilities of the Morehouse School of Medicine. Animals were handled by trained personnel according to Institutional Animal Care and Use Committee guidelines. Mice were initially injected i.p. with monoclonal GDKV-induced aPL Ab in normal saline, at 0 and 48 h, 10 µg mAb per mouse. The control group received equal amount of a murine mAb of irrelevant specificity. The surgical procedure were performed 72 h after the first Ab injection.
Analysis of thrombus dynamics: effects of aPL on thrombus formation. Analysis of thrombus dynamics in a mouse model has been described previously (26, 27, 28). In brief, mice were anesthetized 72 h after the first injection with the aPL or the control IgG, and the right femoral vein was exposed. The vein was pinched with a standard pressure to introduce an injury and to induce a clot. Clot formation and dissolution in the transilluminated vein were visualized with a microscope equipped with a closed-circuit video system (including a color monitor and a recorder). Thrombus size (in square micrometers) was measured when the thrombus reached the maximum size by digitizing the image and tracing the outer margin of the thrombus; the times (in minutes) of formation (from appearance to maximum size) and disappearance (from maximum size to disappearance) of the thrombus were measured as well. Three to five thrombi were successfully induced in each animal, and mean values were computed. Mean thrombus area and mean times for formation, disappearance, and total times were then computed for each group of injected animals. The person performing the surgery and measurements (X.W.L.) was blinded as to what treatment had been given to each animal.
Analysis of endothelial cell activation in the microcirculation of the exposed cremaster muscle of mice. Activation of endothelial cells was assessed by direct visualization and quantitation of adhering ("sticking") WBC to endothelial cells in the microcirculation of the exposed cremaster muscle of mice as described elsewhere (29, 30). In brief, 72 h after the first injection, mice were anesthetized and placed in the dorsal position, the animal’s right scrotum was incised, and the cremaster muscle and testicle were gently exposed and placed on a microscope slide. The evaporative fluid and heat loss was minimized as described previously (29, 30). The dynamic events in the microcirculation of the mouse’s exposed cremaster muscle (thickness, 120 µm only) can be directly visualized without the use of vital dyes. Thus, the lumen of venules and capillaries can be directly viewed and the interaction of individual blood cells (such as WBC, erythrocytes, or platelets) with the luminal surface can be quantitatively assessed. After a stabilization period of 30 min, the leukocytes that remained stationary on the endothelium for at least 30 s were considered adhering ("sticking"). Such adhering WBC were counted under the microscope in five different venules of 25–35 µm diameter; the means were calculated and compared between treated and control groups.
In vitro exposure of endothelial cells to GDKV-induced aPL Abs
Confluent monolayers of HUVEC (104 cells/well) seeded in collagen-coated 96-well plates were incubated with complete RPMI culture medium or GDKV-induced murine monoclonal aPL Abs (neat supernatant) in RPMI culture medium for 4 h at 37°C. As a positive control, some HUVEC monolayers were treated with LPS (3 µg/ml) in complete RPMI for 4 h to increase the surface expression of E-selectin, ICAM-1, and VCAM-1. After paraformaldehyde fixation, adhesion molecule expression was assessed with a colorimetric ELISA previously described (31, 32). Color development was stopped at 3 M H2SO4 at 20 min, and the OD was read at 492 nm wavelength on a SpectraMax 250 ELISA plate reader (Molecular Devices, Sunnyvale, CA). The degree of specific Ag expression was calculated by subtracting nonspecific binding of the secondary Ab from all test values.
Statistical analysis
An independent t test was used to compare the Ab
levels in different groups of mice. The unpaired Student t
test was used to compare the means of thrombus sizes and times (total
disappearance) and adhering WBC numbers between treated and control
groups. Statistical significance was achieved when p 
0.05. Statistically significant differences on surface Ag expression of
endothelial adhesion molecules on HUVEC monolayers exposed to control
medium or to GDKV-induced Abs were evaluated with the unpaired Student
t test. Statistical significance was achieved when
p 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding of ß2GPI to CL-coated plates was
dose dependent and followed a hyperbolic curve. In the presence of 6
µM GDKV, this binding was decreased, which indicates the competition
of GDKV with ß2GPI in binding to CL. The
concentration of ß2GPI required for 50%
saturation increased from 71 nM to 173 nM. Thus, 6 µM GDKV was
equivalent to 102 (173 - 71 = 102) nM
ß2GPI in binding to CL; making it 59-fold
less effective than ß2GPI.
GDKV2 also competed with
ß2GPI in binding to CL in a similar manner, but
with somewhat higher avidity. In the presence of 6 µM
GDKV2, the concentration of
ß2GPI required for 50% saturation increased
from 71 nM to 252 nM. Thus, at 6 µM, GDKV2 was
equivalent to 181 (252 - 71) nM ß2GPI in
binding CL, making it almost 33-fold less effective than
ß2GPI. The percent inhibition of 100 nM
ß2GPI binding to CL by GDKV and
GDKV2 was 43 and 56%, respectively. These
findings confirmed the binding of these peptides to CL, which
was previously reported by other investigators (23, 24).
Direct binding of GDKV peptides to phospholipid
Experiments with mouse antisera to GDKV peptides confirmed the binding of these peptides to CL-coated plates. The optical densitiesfor binding of anti-GDKV to wells containing CL alone and CL+GDKV were 0.322 vs 0.805, and for the anti-GDKV2 were 0.472 vs 0.984. Binding to wells with CL alone indicates aPL activity in mouse sera and the additional binding to wells with CL plus peptide reflects binding of peptides to CL and binding of the antisera to peptides.
Ab production
As expected mice immunized with ß2GPI
produced high levels of aPL in addition to
anti-ß2GPI Abs. Mice immunized with
GDKV-KLH, GDKV-BSA, or GDKV2-BSA, in addition to
producing high levels of Ab against the immunizing peptide and the
carrier proteins, produced significantly (independent t
test: p < 0.05) higher levels of aPL than did mice
immunized with KLH, BSA, free GDKV, or the preimmune control mice (Fig. 2). However, the levels of aPL in mice immunized with whole
ß2GPI were much higher than the aPL levels in
mice immunized with ß2GPI-derived peptides
conjugates. This may be explained in part by the greater affinity of
ß2GPI for PL compared with these peptides
affinity for PL or ß2GPI being more immunogenic
than these peptides. Mice immunized with GDKV-KLH showed significantly
elevated levels of anti-ß2GPI Abs, when
compared with mice immunized with KLH or GDKV alone or nonimmunized
controls (Fig. 2). Similarly, mice immunized with GDKV-BSA and
GDKV2-BSA also had elevated levels of
anti-ß2GPI activity (data not shown). Mice
immunized with BSA or KLH alone showed high levels of Abs to these
proteins (data not shown) together with slightly elevated levels of
aPL, probably due to polyclonal B-cell activation (Fig. 1). However, animals immunized with free
GDKV (with no carrier protein) did not produce any Ab against PL,
ß2GPI (Figs. 1 and 2), or even GDKV (data not shown). The
titration curves for GDKV-induced aPL, and those in mice immunized with
BSA, or KLH, as well as nonimmunized mice are shown in (Fig. 3). Interaction of
ß2GPI with GDKV and inhibition of anti-GDKV
activity by ß2GPI was also determined.
Anti-GDKV was diluted 1:80 in 1% OVA/PBS and incubated with increasing
concentrations of human ß2GPI (12.5–400
µg/ml) for 1 h at room temperature and then overnight at 4°C
before testing by ELISA for anti-GDKV activity.
ß2GPI inhibited the binding of the Ab to the
GDKV-coated plate in a dose-dependent manner, and there was up to 85%
inhibition of anti-GDKV binding by ß2GPI at
400 µg/ml (Fig. 4).
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The GDKV-induced mAb used in the in vivo experiments in addition to anti-GDKV activity had both aPL and anti-ß2GPI activity (mean OD units, 0.565 ± 0.102 and 0.652 ± 0.095, respectively), in preparations containing 550 ng/ml murine Ig. Control murine mAb at similar concentration had negligible aPL and anti-ß2GPI activities: 0.123 ± 0.092 and 0.189 ± 0.098, respectively. Seventy-two hours after the first i.p. injection (immediately before the surgical procedures), mice injected with GDKV-induced aPL showed significantly elevated levels of aPL and anti-ß2GPI Abs (data not shown). Animals injected with control monoclonal preparation were negative for aPL and anti-ß2GPI.
Effect of mAbs on thrombus formation and on activation of endothelial cells in vivo
Mice injected with GDKV-induced aPL Ab produced larger thrombi
that persisted significantly longer when compared with controls
(Table II).
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and 6). This increased adhesion of leukocytes
to endothelial cells is an indication of endothelial cell activation
in vivo.
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As a correlate to the in vivo microcirculation experiments with
GDKV-induced aPL Ab, surface Ag expression of ICAM-1,
E- selectin, and VCAM-1 were quantitated after a 4-h exposure to
GDKV-induced aPL Ab. ELISA analysis revealed that control HUVEC
monolayers constitutively expressed ICAM-1, whereas VCAM-1 and
E-selectin were not detectable (Fig. 7).
However, HUVEC monolayers that were exposed to GDKV-induced aPL Ab for
4 h expressed significantly higher levels of each adhesion
molecule. GDKV-induced aPL Ab was as potent in up-regulating
expression of the adhesion molecules as exposure of HUVEC
monolayers to LPS for 4 h (110–112% of LPS-induced
expression).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrated that induction of aPL is not restricted to immunization with the whole ß2GPI molecule, and peptides representing the PL-binding site of the ß2GPI (GDKV) or peptides with sequences similar to that region that have PL-binding properties (GDKV2) may also induce aPL production. GDKV2 is a hypothetical peptide and is not derived from ß2GPI but has stronger PL-binding properties than GDKV, the ß2GPI-derived peptide. GDKV2 has been used in these studies to show that aPL may be induced by immunization with other PL-binding peptides that are not part of ß2GPI. However, because these PL-binding peptides are too small to be immunogenic by themselves, they need to be coupled to larger protein molecules to become immunogenic. We hypothesize that in vivo a complex of PL-peptide-carrier protein is required for the induction of aPL. In the case of immunization with ß2GPI, which does not need a carrier protein, it is possible to speculate that PL binds to the PL-specific site (GDKV) in the fifth domain of ß2GPI, and the rest of the ß2GPI serves as a carrier protein induces aPL and anti-ß2GPI production.
Similar to the ß2GPI-induced aPL (18, 21), the GDKV-induced aPL were shown to be pathogenic. They caused enlargement of the in vivo induced thrombi in murine femoral veins. The GDKV-induced aPL are associated with anti-ß2GPI activity, and their binding to PL is enhanced by ß2GPI; therefore, it is conceivable that their thrombogenic effect be due to their interference with the regulatory function of ß2GPI on coagulation. The GDKV-induced aPL in this study not only caused enhanced thrombus formation but also caused increased adherence of leukocytes to endothelial cells in vivo. The latter observation may suggest the endothelial cell activation by aPL as a mechanism for their pathogenicity (30, 33).
The levels of aPL induced by ß2GPI were higher than those of aPL induced by GDKV or GDKV2 using the same protocol. This may be explained partly by the greater PL-binding properties of whole ß2GPI compared with these peptides. The other explanation may be the presence of more PL-binding sites other than GDKV on the ß2GPI, that cause formation of larger, PL-containing, and immunogenic complexes with foreign ß2GPI. Another possibility is that the polyclonal Ab responses generated in the mice immunized with ß2GPI would include Abs directed to multiple other epitopes in the intact molecule. In addition, other investigators have observed Abs to other regions of the ß2GPI molecule in patients with aPL. Furthermore, the levels of aPL in patients with APS are never as high as those in animals immunized with ß2GPI, and they are usually more comparable with those seen in mice immunized with PL-binding peptides.
Some investigators have suggested the Id-anti-Id network as a cause for the induction of aPL autoantibodies and APS in mice (18, 34), based on the hypothesis that following immunization with ß2GPI or PL-binding peptide-carrier protein complex Abs against the PL- binding region would be produced that will be similar to PL, and anti-idiotypic Abs against them may have anti-PL activity.
Not all PL-binding proteins are capable of aPL induction by immunization, and it is likely that PL may need to bind in some specific way to form an immunogenic, aPL-inducing complex. For example, placental anticoagulant I (annexin V) which binds PL very avidly and much more effectively than ß2GPI does not induce aPL production by immunization and induces only anti-annexin V in animals (35).
Altogether, these data indicate that immunization with foreign ß2GPI, or its PL-binding site, can induce high levels of pathogenic aPL. However, it is unlikely that this would be the cause of aPL production in patients with APS, but incidental exposure to other PL-binding foreign proteins such as viral or bacterial products, with structural and functional similarities to the PL-binding site of ß2GPI may induce pathogenic aPL and anti-ß2GPI production and cause APS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Azzudin Gharavi, Department of Medicine, Morehouse School of Medicine, 720 Westview Drive, S.W., Atlanta, GA 30310-1495. E-mail address:
3 Abbreviations used in this paper: aPL, antiphospholipid Ab; APS, antiphospholipid syndrome; ß2GPI, ß2-glycoprotein I; PL, phospholipid; CL, cardiolipin; EC, endothelial cell; WBC, white blood cell; KLH, keyhole limpet hemocyanin; SCR, short consensus repeat.
Received for publication December 3, 1998. Accepted for publication June 15, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), or KLH (
), as well as the nonimmunized
mouse sera (
) were serially diluted in 10% ABS and tested by ELISA
for aPL activity.
