This review is part of a thematic series on Transplant Vasculopathy, which includes the following articles:
Allograft Vasculopathy Versus Atherosclerosis
Antibody and Complement in Transplant Vasculopathy
Cytokines, Interferon-␥, and Transplant Vasculopathy
Chemokines and Transplant Vasculopathy
Stem Cells and Transplant Vasculopathy
William M. Baldwin III and Jordan Pober, Guest Editors
Antibody and Complement in Transplant Vasculopathy
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Jennifer Wehner, Craig N. Morrell, Taylor Reynolds, E. Rene Rodriguez, William M. Baldwin III
Abstract—Advances in immunosuppression have decreased the incidence of acute rejection, but the development of
vasculopathy in the coronary arteries of transplants continues to limit the survival of cardiac allografts. Transplant
vasculopathy has also been referred to as accelerated graft arteriosclerosis because it has features of arteriosclerosis, but
it is limited to the graft and develops over a period of months to years. Although the pathological features of transplant
vasculopathy are well defined, the causative mechanisms are not completely understood. This review focuses on the
mechanisms by which antibody and complement can cause or contribute to coronary vasculopathy in cardiac transplants.
Antibodies and complement can have independent effects, but the combination of antibodies and complement with
inflammatory cells has greater pathogenic potential for the endothelial and smooth muscle cells of the coronary arteries.
For example, stimulation through receptors for IgG or complement split products can activate macrophages, but
stimulation through combinations of these receptors generates synergistic results. Together, antibodies and complement
efficiently integrate the activation of endothelial cells, platelets, and macrophages, which are 3 of the primary
components in the pathogenesis of transplant vasculopathy. Recent findings indicate that antibodies and complement
produced within the transplant may contribute to vascular pathology in some transplants. Acute rejection caused by
antibodies and complement has been treated by combinations of plasmapheresis, intravenous ␥ -globulin and
monoclonal antibodies to CD20 on B lymphocytes. The effect of these treatment modalities on the development of
coronary vasculopathy is unknown. (Circ Res. 2007;100:191-203.)
Key Words: antibodies 䡲 complement 䡲 platelets 䡲 macrophages 䡲 coronay arteries
T
mental models have been a major source of current concepts
of the pathogenesis of the vascular changes (Figure 1B).
Although the progression of vasculopathy in transplants can
be accelerated by nonimmunological risk factors, such as
hypertension and hyperlipidemia (reviewed for this thematic
series by McManus and colleagues1), alloimmune responses
are thought to initiate the vascular pathology because these
vascular lesions are much more readily produced in experimental allografts than isografts and in normal than immune-
ransplant vasculopathy culminates in diffuse concentric
intimal expansion and adventitial sclerosis of the coronary arteries (Figure 1A). Surveillance angiographic studies
demonstrate narrowing of the lumen of the coronary arteries
and their major branches by this process before pathological
changes can be observed in the small arterioles that are
captured in endomyocardial biopsies. Therefore, direct pathological observations of the initial stages of transplant vasculopathy are limited in human hearts, and data from experi-
Original received September 24, 2006; revision received November 24, 2006; accepted December 1, 2006.
From the Departments of Pathology (J.W., T.R., W.M.B.) and Comparative Medicine (C.N.M.), Johns Hopkins University School of Medicine,
Baltimore, Md; and the Department of Pathology (E.R.R.), Cleveland Clinic Foundation, Ohio.
This manuscript was sent to Bradford C. Berk, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to William M. Baldwin III, MD, PhD, Department of Pathology, Ross Research Bldg, Rm 659, The Johns Hopkins University School
of Medicine, 720 Rutland Ave, Baltimore, MD 21205-2196. E-mail [email protected]
© 2007 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000255032.33661.88
191
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February 2, 2007
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Figure 1. Histology of transplant vasculopathy. A, Epicardial
coronary artery from a human cardiac transplant with diffuse
concentric intimal expansion (i) and adventitial sclerosis (ad). B,
Intramyocardial artery from a rat cardiac allograft with transplant
vasculopathy. Endothelial cells are stained black by immunoperoxidase for a complement split product (C4d).
deficient recipients. In these experimental models, immunemediated injury of the endothelial cells results in infiltrates of
mononuclear cells and proliferation of smooth muscle cells
that expand the intima. There is experimental evidence that
both T lymphocytes and antibodies can contribute to the
initial vascular injury. Although the intimal and adventitial
pathology contain mononuclear cell infiltrates, the mediators
found in the intima and adventitia are different.2 This review
focuses on the mechanisms by which antibody and complement can cause or contribute to myointimal expansion in the
large arteries of cardiac transplants. Antibodies and complement are particularly relevant to transplant vasculopathy
because receptors for antibodies and complement are expressed by all of the cells found in the lesion. Macrophages,
neutrophils, eosinophils, natural killer (NK) cells, B lymphocytes, platelets, and endothelial cells express a range of
receptors for the Fc tail of antibodies as well as for various
complement components.
Antibody Responses to Transplants
Transplants are powerful stimulants of antibody production.3
The full impact of antibodies on transplants was manifested
in the early years of renal transplantation. The most devastating effect of antibodies is hyperacute rejection, in which
the transplant is rejected within minutes to hours after
circulation is reestablished to the grafted organ.4 Hyperacute
rejection is characterized by deposits of antibodies and
complement components on the blood vessels associated with
extensive disruption of the vasculature, as evidenced by fibrin
deposition and hemorrhage. Before 1969, antibodies stimulated by a first transplant frequently led to hyperacute
rejection of a second renal transplant. However, in 1969,
Patel and Terasaki5 described a crossmatch test that detected
antibodies to prospective donors in sera from potential
transplant recipients. Since the introduction of crossmatch
testing, hyperacute rejection has been almost eliminated.
The capacity of organ transplants to elicit antibody responses was more obvious when azathioprine and steroids
were the mainstay of clinical immunosuppression. In that era,
antibody responses were not controlled effectively and rejection was frequently characterized by immunoglobulin and
complement deposits in blood vessels of transplants.6,7 As
increasingly effective immunosuppressive protocols were
developed, overt evidence of immunoglobulin deposits in
transplant biopsies was diminished and attention to antibodies
waned. However, more sensitive techniques for measuring
circulating antibodies in the serum and complement deposition in biopsies have demonstrated that antibodies and complement continue to be associated with acute and chronic
rejection. Antibody responses continue to present practical
clinical problems because many patients have been exposed
to histocompatibility antigens by previous blood transfusions,
transplants, or pregnancy. Prior exposure to histocompatibility antigens results in memory B and T lymphocytes that are
more easily stimulated by antigen, and, as a consequence, are
less susceptible to immunosuppression. Moreover, some of
the current protocols that attempt to reduce immunosuppression to a single agent are compromised by unexpectedly high
incidences of antibody responses.8
Antibody responses to transplants are not monitored routinely in transplant recipients, but one large multicenter study
found that the frequency of antibodies to human leukocyte
antigen (HLA) was 22.8% in 393 recipients of cardiac
transplants, which was similar to the frequency of 20.9%
found in renal transplant recipients.9 This estimate of antibody responses is likely to be low because the prevalence of
antibodies in clinical transplant recipients is underestimated
by routine serological techniques. Assays using isolated HLA
or recombinant HLA for ELISA or flow-bead platforms can
detect antibodies that are below the threshold routine serological assays.10 Even the application of these more sensitive
assays for antibodies in serum measures only the unbound
alloantibodies in the blood compartment and fails to detect
antibodies bound to target antigens in the graft. Usually more
antibodies can be eluted from rejecting transplants than can
be found in the circulation.11–13 Elution studies are of more
value in acute than chronic rejection because antibodies do
not bind irreversibly to antigens. Antibodies either release or
are shed from the surface of cells, depending on their avidity
of binding. As a result, antibodies can initiate injury and then
depart by the time transplant vasculopathy is diagnosed.14
In experimental transplants to animals treated with minimal immunosuppression, antibodies and complement can be
Wehner et al
Antibody and Complement in Transplant Vasculopathy
193
TABLE 1. Chemokines, Adhesion Molecules, and Growth Factors Expressed by Endothelial Cells in
Response to Both Antibody and Complement
Function
Mediators
Stimulants
Adhesion
von Willebrand factor, P-selectin
MHC crosslinking by antibody; C5a or MAC stimulation of
endothelial cells
MCP-1 (CCL2), IL-8 (CXCL8), RANTES (CCL5)
MHC crosslinking by antibody; C1q, C3a, C5a, or MAC
stimulation of endothelial cells
Platelet-derived growth factor, basic FGF
MHC crosslinking by antibody; MAC stimulation of
endothelial cells
Chemotaxis
Growth Factors
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demonstrated bound to endothelial cells in the large arteries
as well as capillaries and veins early after transplantation.15 In
contrast, the infiltrates of T lymphocytes are concentrated
around capillaries and in periarterial adventitial spaces.16
These histological findings reflect the fact that antibodies are
the product of a selection process that results in high-affinity
binding. High-affinity antibodies are capable of binding to
vascular endothelial cells in the high flow conditions of large
arteries. Russell and colleagues17,18 have demonstrated the
capacity of passively transferred alloantibodies to cause
vasculopathy in arteries of hearts transplanted to immunosuppressed or immunodeficient mice. Together, these experimental models establish the capacity of antibodies to bind to
the endothelium of large arteries and to induce pathological
lesions. Obviously, these experiments do not exclude a role
for cell-mediated immune responses, and many of the mechanisms of antibody-mediated injury are dependent on cells.
Each subsequent section below highlights mechanisms by
which antibody and complement activate vascular tissue as
well as leukocytes.
Fab fragments do not cause exocytosis, but bivalent F(ab⬘)2
fragments are effective in triggering exocytosis. The multimeric von Willebrand factor that is released from the Weibel–
Palade granules is effective at aggregating platelets even in
the high shear stress conditions of arteries. In contrast to von
Willebrand factor, P-selectin has low affinity for ligands on
leukocytes that results in transient interactions in the lowflow conditions of capillaries and venules. In the high-flow
environment of arteries, P-selectin may act as a signaling
molecule between endothelial cells or platelets and monocytes enmeshed in von Willebrand factor.21–23 As discussed
below, platelet activation could be an important early stage in
the development of vasculopathy because platelets release
chemokines and express the costimulatory molecule CD154
for lymphocytes, macrophages, dendritic cells, and endothelial cells.24
Crosslinking of Major Histocompatibility
Complex Antigens Antibodies Causes Rapid
Exposure of Adhesion Molecules by
Endothelial Cells
The rapid release of preformed molecules from Weibel–
Palade storage granules in endothelial cells is followed by a
more protracted synthetic response that includes upregulation
of chemokines and growth receptors. Mouse endothelial cells
produce monocyte chemoattractant protein-1 (MCP-1;
CCL2) after stimulation with antibodies to MHC class I
antigens.25 This chemokine recruits monocytes and macrophages to vascular endothelium. Macrophages attracted by
MCP-1 have receptors for the Fc tail of antibodies (FcR).
Through these receptors, antibodies bound to endothelial cells
can engage and activate neutrophils and macrophages.
Bian and Reed have demonstrated that crosslinking HLA
by antibodies signals upregulation of receptors for fibroblast
growth factor (FGF) by endothelial cells and smooth muscle
cells.26 The expression of FGF receptor contributes to the
proliferation of smooth muscle cells caused by antibodies to
HLA.
It is important to note that these experiments on endothelial
responses to antibodies have all been confirmed with purified
monoclonal antibodies because Rose and colleagues27 demonstrated that interleukin-1 (IL-1) can contaminate antibodies
prepared from human serum. IL-1 was found to be responsible for the upregulation of adhesion molecules on endothelial
cells that had been attributed to alloantibodies.
As discussed in subsequent sections, these responses to
antibodies are reinforced by activation of complement because isolated complement components can induce immedi-
The antibodies that have been correlated most closely with
vascular pathology in transplants are antibodies to antigens of
the major histocompatibility complex (MHC). Arterial endothelial cells normally express high levels of MHC class I
antigens in experimental animals and in humans. The expression of MHC class II antigens is less universal on endothelial
cells. In humans, unlike rodents, capillary and venous endothelia express MHC class II antigens constitutively. MHC
class II antigens are not expressed on most quiescent arterial
endothelia in animals or humans, but human coronary arteries
are exceptions.19 In addition, expression of MHC class II
antigens can be upregulated by many inflammatory mediators, most notably interferon-␥ (IFN-␥). Upregulation of
MHC class II antigens is a recognized consequence of acute
rejection episodes.
Antibodies to human MHC antigens can have multiple
direct and indirect effects on endothelial cells. IgG antibodies
are bivalent and can crosslink MHC antigens. As a result, IgG
antibodies to HLA class I antigens stimulate a rapid release of
preformed adhesion molecules, such as von Willebrand factor
and P-selectin, from the Weibel–Palade storage granules in
endothelial cells (Table 1).20 Crosslinking of HLA by antibody is required to activate exocytosis because monovalent
Protracted Synthetic and Proliferating
Responses of Endothelial and Smooth Muscle
Cells to Crosslinking of MHC Antigens
by Antibodies
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February 2, 2007
ate release of the same preformed adhesion molecules from
Weibel–Palade bodies followed by the synthesis of mediators
and proliferation of endothelial cells (see Table 1).
Antibodies to Antigens Induced by Activation
of Endothelial Cells
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Additional antigenic targets for antibodies can be generated
on injured or activated endothelial cells. Autoantigens that are
cryptic in normal endothelial cells can be exposed as the
result of granzyme B released from lymphocytes. Rosen and
colleagues28 have demonstrated that granzyme B cleaves
autologous proteins to expose cryptic epitopes that become
targets of autoantibodies. Antibodies to several autoantigens
have been correlated with transplant vasculopathy. For example, antibodies to the protein filament vimentin have been
demonstrated in nonhuman primate and human studies of
cardiac transplantation.29,30 Vimentin, which is not normally
expressed on endothelial cells, is present on apoptotic cells.
Complement deposition (in the form of C4d) has been found
in arteries of cardiac allografts to cynomolgus monkeys with
high titers of IgM antibodies to vimentin, even in the absence
of alloantibodies. Antibodies formed in cardiomyopathies
before transplant may also play a role in graft rejection. High
titers of preformed IgG antibodies to cardiac myosin have
been correlated with decreased cardiac graft survival at 2
years.31 In mice, antibodies to myosin accompany acute and
chronic rejection.32,33
All of these studies suggest that an initial insult to the graft
may lead to an increased range of antigens to expand the
antibody responses.
Figure 2. Fc and complement receptors on macrophages. Macrophages can express 5 different receptors (4 activating and 1
inhibitory) for the Fc tail of IgG as well as receptors for C1q,
C4b, C3b, and iC3b. The relative amount of different Fc and
complement receptors expressed is determined by the activation status of the macrophage. Stimulation through receptors for
C5a or Fc␥RIII causes a reciprocal upregulation.
Activation of Leukocytes Through FcR
by Antibodies
expressed by B lymphocytes and functions as a feed back
inhibitor of antibody production. The balance of activating
and inhibitory FcR expression by macrophages is controlled
by different mediators including cytokines and complement
split products. For example, IFN-␥ modulates the balance of
receptor expression by upregulation of Fc␥RI and Fc␥RIII
and downregulation of Fc␥RIIB.
Although antibodies can activate cells solely through FcR,
the simultaneous stimulation through FcR and complement
receptors can cause synergistic effects. Therefore, a consideration of complement is relevant before the effects of FcR
are considered in further detail.
Regardless of specificity, once bound to antigen, the Fc tail of
antibodies can interact with FcR on cells. The responses of
neutrophils and macrophages to antibodies depend on the
type of FcR engaged and concurrent signals from other
receptors, such as receptors for different complement split
products (Figure 2). Macrophages express the high-affinity
Fc␥RI (CD64), which is capable of binding monomeric IgG,
as well as the low-affinity Fc␥RIIA (CD32), Fc␥RIII (CD16),
and Fc␥RIV, which are engaged by clusters of IgG on
antigen.34 These Fc␥R signal through immunoreceptor
tyrosine-based activation motifs (ITAMs). Activation of macrophages through FcR upregulates many proinflammatory
functions, including the production MCP-1 and IL-8 (Table
1), and promotes their survival at sites of inflammation.35,36
Fc␥RIII is expressed by NK cells as well as macrophages,
and this receptor signals through ITAM to initiate antibodydependent cell-mediated cytotoxicity (ADCC) by NK cells.
Because NK cells are rich sources of granzyme B, engagement of NK cells can diversify the antigenic stimulation to
include autoantigens. Endothelial cells and platelets also
express Fc␥RIIA, but the function of this receptor on these
cells is less well understood. This is an area of great relevance
to potential pathology in organ transplants.
Activating Fc receptors are counterbalanced by Fc␥RIIB
on macrophages, which functions through an immunoreceptor tyrosine-based inhibitory motif (ITIM). Fc␥RIIB is also
In addition to the direct effects that antibodies can have
through crosslinking antigens and engaging FcR on leukocytes, certain subclasses of antibodies can alter endothelial
cell and leukocyte function through activation of the complement system. Complement is a multifunctional system of
receptors and regulators as well as effector molecules. The
pathogenic power of complement is based on the capacity of
the complement system to amplify both innate and adaptive
immunity. This amplification is accomplished through 2
strategies. First, enzymatic reactions in the complement
cascade generate multiple potent biological split products.
Second, these split products stimulate leukocytes, platelets,
and parenchymal cells through either specific receptors or
receptor-independent pore formation (Figure 3).
Antibodies provide binding sites for C1 that initiates the
enzymatic cascade of the classical pathway of complement.
Bound C1 can enzymatically cleave C4 and C2, which then
form an enzyme complex that is capable of cleaving C3.
Amplification occurs because multiple C4 and C3 molecules
are cleaved and each C4 and C3 produces 2 biologically
active fragments: both C4 and C3 produce small soluble
mediators of chemotaxis and activation (C4a and C3a), and
large fragments that can bind covalently to proteins or
carbohydrates (C4b and C3b). C3b has the potential to
Activation of Complement by Antibodies
Wehner et al
Antibody and Complement in Transplant Vasculopathy
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Figure 3. Phases of complement activation. A, Activation of
complement progresses through initiation, amplification, regulation, and terminal complex formation. Antibodies bound to antigens on the transplant initiate the complement system via C1.
Amplification occurs through a succession of enzymatic steps
that split many C4 and even more C3 molecules. Continuous
enzymatic cleavage is prevented by a number of RCA. Regulation of C4 and C3 limits amplification, but small amounts of C3b
can perpetuate the complement cascade when it forms an
enzymatically active C5 convertase that can cleave C5. Cleavage of C5 results in 2 biologically active split products, C5a and
C5b. C5b binds to C6 through C9 to form the MAC. Leukocytes
have receptors for fluid phase (C4a, C3a, and C5a) and tissuebound (eg, C4b and C3b) complement split products as well as
the Fc portion of antibodies. B, Regardless of the mechanism
by which C3b is deposited on a cell, factor B of the alternative
complement pathway can bind to C3b. Cleavage of factor B by
factor D produces a C3 convertase (C3bBb) that cleaves more
C3. This autocrine activation of C3 is known as the amplification
loop.
expand complement activation further because once it is
bound to tissues, C3b can complex with factor B of the
alternative pathway and initiate a self-perpetuating amplification loop that results in more C3b deposition (Figure 3B).
In inflammation, these enzymatic reactions result in exponential increases in C4 and C3 split products. One study
calculated that each C1 caused approximately 25 C4b and
250 C3b molecules to be bound to a cell surface.37 The
amplification caused by C4b and C3b is not only quantitative;
it is also kinetic. The ability of C4b and C3b to form covalent
bonds with tissue results in a longer half-life for these
complement components than for antibodies, C1 or C2, all of
which can be shed rapidly.15 The amplification of complement in transplants is reflected by the fact that C4b and C3b
195
split products are more readily detectable than antibodies in
transplant biopsies even when relatively high titers of antibodies are present in the circulation.
Antibodies are the prime cause of complement activation
in the high-flow conditions in the lumen of the coronary
arteries, but 2 additional pathways of complement activation
can also contribute to vascular pathology. The lectin pathway
is initiated through mannose binding lectin (MBL). Similar to
C1, bound MBL can activate the complement cascade by
cleaving C4. MBL has globular lectin ends that can bind to
terminal sugars on bacteria but also to injured or apoptotic
endothelial cells.38 MBL participates in ischemia/reperfusion
injury to hearts,39 and it may also be activated by cells
undergoing apoptosis in transplants.
The third means of complement activation is the alternative
pathway. This pathway has relevance to transplants both in
the absence and presence of antibody. As mentioned above,
factor B can bind to C3b that has been deposited on tissues as
the result of antibody activating C1. In this capacity, the
factors of the alternative pathway (factor B, factor D, and
properdin) act as an autocrine amplification loop to generate
more C3b. This mechanism of amplification can be responsible for the majority of C3b generated.40 The alternative
pathway can also be initiated by spontaneous binding of small
amounts of C3 to ischemic tissues. Unlike the classical and
lectin pathways, which are triggered by particular substrates
such as antibodies or phospholipids exposed on the plasma
membrane, the alternative pathway is dependent on sporadic
spontaneous conformational change in C3 at a rate of approximately 1% per hour. This mechanism is now thought to be of
importance in the pathogenesis of ischemia/reperfusion41 as
occurs to the organ at the time of transplantation.
The amount of C3b bound to proteins is influenced by the
properties of the available proteins and of the surrounding
membrane structure. The available binding sites for C3b may
be significantly increased in vessels of transplants when
platelets become attached and activated on the endothelial
cells because P-selectin on activated platelets has been found
to bind C3b efficiently.42 This would result in a concentration
of P-selectin and C3b deposits on endothelial cells to stimulate leukocytes.
Once C3b is deposited on tissues by any or a combination
of these activation pathways, C3b can propagate the complement cascade when it forms an enzymatically active C5
convertase that cleaves C5 into 2 biologically active split
products, C5a and C5b. The smaller split product, C5a, is an
extremely potent mediator of chemotaxis and activation for
leukocytes; the larger split product, C5b, binds to C6 through
C9 to form a tubular structure, termed the membrane attack
complex (MAC), that is capable of creating a pore in plasma
membranes. Large amounts of MAC can disrupt the vascular
endothelial cell surface by lysis, but sublytic numbers of
MAC can cause activation of endothelial cells as discussed
subsequently. Except in cases of hyperacute rejection, endothelial cell activation is found more frequently than lysis.
Traditionally, activation of C5 has been thought to be
dependent on pathways causing activation of C3, but there are
now many models in which C5 is activated independently of
C3. Serine proteases, such as thrombin, are known to cleave
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C5, and recently this mechanism has been demonstrated to be
upregulated in C3 knockout mice.43 Gessner and colleagues44,45 have found that production and cleavage of C5 by
macrophages is regulated by FcR. These researchers have
demonstrated a feedback loop between FcR and C5a in
several models of autoimmunity. In these models, C5 synthesis and cleavage is upregulated by antibodies engaging
Fc␥RIII. Binding of the resultant C5a to receptors on macrophages in turn upregulates Fc␥RIII and downregulates the
inhibitory Fc␥RIIB. This feedback is independent of the early
components of complement because it occurs in C3 knockout
mice. In the absence of C3, C5 is cleaved by an uncharacterized enzyme produced by macrophages. It is likely that this
mechanism is even more significant in normal individuals
because macrophages can synthesize all of the complement
components, and therefore, macrophages could generate
more C5a through multiple pathways including the alternative pathway. The large numbers of macrophages infiltrating
arteries of transplants would provide a rich environment for
macrophages to propagate inflammation through complement. We have demonstrated that macrophages can be a
critical source of complement in acute rejection of cardiac
allografts.46 Local complement production also has been
documented in conventional atherosclerotic plaques.47
Regulators of Complement Activation
Uncontrolled amplification of complement activation by continuous enzymatic cleavage would consume all of the complement effector molecules and result in extensive tissue
damage. These potentially deleterious effects of complement
amplification are limited by multiple regulatory molecules.
Of particular relevance to vascular pathology is the group
of regulators that has evolved to control the exponential
generation of split products from the structurally related C4b
and C3b components. This group of 6 regulators of complement activation is encoded in the RCA (Regulators of
Complement Activation) gene cluster that includes decay
accelerating factor (DAF; CD55), membrane cofactor protein
(MCP; CD46), complement receptor type 1(CR1; CD35),
complement receptor type 2 (CR2; CD21), C4 binding
protein (C4bp), and factor H. The first 4 regulators are
expressed as membrane bound proteins, and the last 2
circulate in the plasma. DAF and MCP are present on a wide
variety of cells including leukocytes and vascular endothelial
cells, whereas CR1 and CR2 are expressed on selected
leukocytes and antigen presenting cells.
Although these regulators are structurally related, they
work by 2 different processes (Table 2). One set works by
separation of associated complement components (eg, dissociation of C4b and C2a by DAF), and the second set (eg,
MCP) function as cofactors for factor I, allowing it to cleave
C4b and C3b into biologically active fragments: first iC4b or
iC3b and then C4dg or C3dg. CR1 is remarkable because it
regulates both as a dissociating factor and as a cofactor. These
regulatory mechanisms truncate the expansion of the complement split products by disrupting the enzymatic activity, but
a resulting succession of fragments remain attached to the
membrane and serve as ligands. For example, C3b, iC3b, and
C3d serve as ligands for cells with complement receptors
TABLE 2.
Function of Complement Regulatory Proteins
Classical or MBL (C4bC2a)
Alternative (C3bBb)
C4bp
Factor H
DAF*; CR1†
DAF*; CR1†
Dissociation
Soluble
Membrane
Cleavage
Enzyme
Factor I
Factor I
Cofactor
CR1†; MCP*
CR1†; factor H
*Decay accelerating factor (DAF; CD55) and membrane cofactor protein
(MCP; CD46) widely expressed on leukocytes and parenchymal cells. These
molecules also modulate T-lymphocyte responses. †Complement receptor 1
(CR1; CD35) expressed on leukocytes.
CR1, CR3, and CR2, respectively (see Figure 2). As a result,
complement-mediated damage in transplants is characterized
by accumulation of neutrophils and macrophages,48,49 which
possess CR1 and CR3.
Even when significant amounts of C4 and C3 fragments
are deposited in normal autologous or allogeneic tissues,
relatively little MAC is formed because of the density and
diversity of regulators directed at the components of C4 and
C3 (Figure 3). Lysis of autologous or allogeneic cells is
further inhibited by regulatory proteins, such as CD59, that
inhibit MAC formation. Although these regulators usually
avert lysis of allogeneic cells, insertion of sublytic quantities
of MAC into leukocytes or parenchymal cells can cause
activation and promote inflammation.50 –55
Expression and protection of tissues by RCA does vary.
Basal levels of RCA vary for different cells, and these levels
can be modulated by inflammation and tissue injury. Many
mediators that have been implicated in acute and chronic
responses to transplants regulate DAF expression by endothelial cells in vitro. Mediators of acute inflammation, such as
thrombin, tumor necrosis factor-␣ (TNF-␣), and IFN-␥,
increase DAF expression but not CD59 or MCP expression.56,57 The effects of cytokines on DAF expression is
augmented in endothelial cells with MAC deposited on their
surface. MAC upregulation of DAF expression may represent
a feedback mechanism to protect vascular integrity in sites of
complement activation.
Mediators associated with chronic inflammation, such as
basic FGF and vascular endothelial growth factor also upregulate the expression of DAF. The finding that cyclosporine
A inhibits the upregulation of DAF expression suggests that
some of the mechanisms that protect tissues from complement activation may be inhibited in transplant patients.58
Therefore, the amount of complement injury in transplants
is likely to be dependent on both the level of complement
activation and the expression of complement regulators.
Decreased or inhibited expression of complement regulators
would promote vascular injury, whereas upregulation of
complement regulators could protect transplants from continued antibody responses. There are in vitro and in vivo data
that complement regulators can be upregulated to protect
endothelial cells from acute antibody-mediated rejection as
part of a process called accommodation.59 The expression of
complement regulators is under investigation in endomyocar-
Wehner et al
Antibody and Complement in Transplant Vasculopathy
dial biopsies and coronary arteries in transplants with transplant vasculopathy.
C-Reactive Protein Interactions
With Complement
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C-reactive protein (CRP) is another recognition pathway that
has been implicated in activating complement. As an acutephase reactant, large amounts of CRP are synthesized rapidly
in response to tissue injury and inflammation.60 CRP binds to
phosphatidylcholine and sphingomyelin that are exposed on
plasma membranes of injured cells. After CRP complexes, it
can engage the classical pathway of complement by binding
C1q.61,62 CRP simultaneously regulates as well as activates
complement because CRP also binds factor H. Factor H limits
the amplification loop and acts as a cofactor for factor I to
cleave C3b to iC3b. Although iC3b is a ligand for CR3,
stimulation through CR3 may deviate the immune response
by downregulating IL-12 production by human monocytes.63
In addition to marking apoptotic cells with iC3b, CRP may
interact directly with Fc␥RI and Fc␥RIIA on macrophages.64
On the whole, therefore, CRP may serve to clear injured
tissue by noninflammatory means.
Complement Activation of Vascular
Endothelial, Smooth Muscle Cells,
and Fibroblasts
Endothelial cells are responsive to many components of
complement activation. Human vascular endothelial cells and
smooth muscle cells are stained by antibodies to C5a receptor.65 The potential for tissue activation through C5a receptors
has been verified for endothelial cells in culture. Foreman et
al66 found that C5a can activate endothelial cells to release
von Willebrand factor and P-selectin. Interaction of C5a with
its receptor, a 7-membrane spanning molecule, results in
intracellular signaling. This receptor is sensitive to pertussis
toxin and is coupled to G1 in both leukocytes and endothelial
cells. However, the signal cascade that results from ligation
of the C5a receptor on endothelial cells is distinct from that
on leukocytes. Unlike C5a-mediated leukocyte chemotaxis,
C5a mediated endothelial cell chemotaxis, as well as cell
retraction and actin polymerization, depends on transactivation of the epithelial growth factor receptor.67 Activation of
endothelial cells by C5a is augmented by proinflammatory
cytokines, such as TNF-␣. Leukocyte chemotaxis is augmented by C5a induction of IL-8 (CXCL8) and RANTES
(CCL5) production by endothelial cells.68 IL-8 can have
additional effects on endothelial cells including increased
survival and proliferation.69
Through data mining, Mastellos et al70 found that C5a has
been linked to the following functions: chemotaxis, cell
migration, cell invasion, endothelial cell activation, and cell
proliferation, among many others. All of these functions
could contribute to the stromal component of the lesion in
chronic vasculopathy. C5a could potentially influence the
accumulation of the poorly differentiated smooth muscle cells
within the neointima, whether they are derived from the
media or from adventitial fibroblasts or from circulating host
derived precursors, smooth muscle cells, or mesenchymal
197
stem cells. The possibility that C5a causes smooth muscle
cells or their progenitors to migrate remains to be tested.
A contribution of C5a to the adventitial fibrosis is also
possible. C5a has been implicated in fibrocyte dedifferentiation and fibroblast proliferation in models of pulmonary
fibrosis. In chronic bleomycin-induced pulmonary fibrosis,
C5 is associated with expression of matrix
metalloproteinase-3 and transforming growth factor-␤1.71
Similarly, hepatic stellate cells differentiate into myofibroblasts and increase their expression of C5a receptor in
response to liver injury. These observations suggest that it
would be fruitful to explore the generation of C5a and the
expression of C5aR within transplant vasculopathy lesions.
Additionally, small molecule inhibitors of C5a could be
useful as experimental and therapeutic tools in transplant
vasculopathy. Hillebrandt et al72 found that small molecule
inhibitors of C5a decreased production of collagen and
hepatic fibrosis in genetically susceptible mice. The potential
for inhibitors of C5a to decrease migration of vascular
smooth muscle into the neointima is worth exploring.
Endothelial cells also express receptors for C1q and C3a.
The responses of endothelial cells to these complement
components need to be studied more completely. It is known
that C1q stimulates human endothelial cells to produce the
chemokines IL-8 and MCP-1 in vitro.73 C3a, like C5a,
upregulates IL-1␤ in addition to the chemokines IL-8 and
RANTES. The stimulation of this set of mediators (IL-8,
MCP-1, and RANTES) is a shared pathway of multiple
functions of antibody and complement activation (Table 1).
Therefore, chemoattraction of neutrophils and macrophages
would be one major effect of complement activation on
arterial endothelium. Indeed, antibody activation of complement in cardiac transplants is associated with subendothelial
accumulation of macrophages in capillaries and
arteries.15,16,74,75
Chemotaxis and adhesion is intensified by insertion of
sublytic amounts of MAC into the plasma membrane of
endothelial cells and smooth muscle cells. The immediate
effect of purified C5b-C9 on endothelial cell cultures is the
translocation of P-selectin and von Willebrand factor from
Weibel–Palade storage granules to the plasma membrane.50
von Willebrand factor ensnares platelets and monocytes
against the endothelium, and P-selectin costimulates their
production of inflammatory mediators.21,22 Intravascular aggregation of P-selectin– and von Willebrand factor– expressing platelets is a prominent feature of experimental models of
antibody-mediated rejection that is decreased in the absence
of MAC deposition.46,76
In addition to releasing preformed mediators, MAC-activated endothelial cells synthesize IL-8, MCP-1, P-selectin,
tissue factor, platelet-derived growth factor and basic
FGF.77,78 The secretion of basic FGF may result in autocrine
stimulation when antibodies to HLA cause upregulation of
FGF receptors on endothelial cells.26 In vitro studies have
demonstrated that MAC can have direct proliferative effects
on vascular smooth muscle cells, as well as decrease apoptosis of these cells.79 These effects would strengthen the
proliferative and survival signals induced by antibodies to
HLA. The relevance of the effects of MAC on transplant
198
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February 2, 2007
vasculopathy has been verified in cardiac transplants to
C6-deficient rats. The C6 deficiency prevents MAC formation and slows the progression of vasculopathy in cardiac
transplants.80
Platelets Can Potentiate Vasculopathy
Through Complement Activation
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The localization of platelets on arterial endothelium through
P-selectin and von Willebrand factor can augment innate and
adaptive immune responses in the vessel. Activated platelets
augment recruitment of leukocytes by both secreting chemokines and inducing endothelial cells to secrete chemokines.
Platelets secrete IL-8, RANTES, MIP-1a (CCL3), and
MCP-3 (CCL7), which recruit leukocytes. Endothelial cells
stimulated by platelets secrete MCP-1 and IL-8. The finding
that P-selectin on activated platelets can bind C3b and
propagate the complement cascade provides more routes for
recruitment and activation of leukocytes. C1q, MBL, and
CRP can enter the equation when phospholipids are exposed
on the plasma membranes of activated platelets. C1q, MBL,
and CRP can bind to phospholipids, such as phosphatidylserine. Thus, a carpet of activated platelets would enrich and
extend the area available for complement interactions.
Platelets not only potentiate the localization of leukocytes
to arteries; they can also activate leukocytes of both the innate
and adaptive immune system. The expression of P-selectin on
platelets provides costimulation to monocytes for the production of inflammatory mediators including MCP-1 and TNF␣.21,22 This function of P-selectin may be greatly augmented
by the activation and binding of C3b to P-selectin. In
addition, platelets are a major source of soluble CD154,
which is the ligand for CD40 on B lymphocytes, macrophages, dendritic cells, and endothelial cells.24 Plateletderived CD154 has been demonstrated to initiate rejection of
hearts transplanted to CD154-deficient mice.81 Finally, platelets secrete a variety of growth factors, such as plateletderived growth factor, endothelial growth factor, and FGF,
that promote proliferation of endothelial cells, smooth muscle
cells, and fibroblasts.82
Even though the contribution of platelets to atherosclerosis
has been studied extensively, immunologists have not considered the potential contributions of platelets to transplant
vasculopathy.
Chemoattraction and Activation of
Macrophages by Complement
Macrophages have a range of receptors for Fc and complement split products. C3a and C5a mediate chemotaxis and
activation of macrophages through C3aR and C5aR. C5a is
quantitatively much more potent as a chemotactic agent than
C3a. Activation of monocytes through their C5a receptor
causes upregulation of CR1 and CR3 and production of
proinflammatory cytokines, such as IL-1, IL-6, IL-8, and
TNF-␣.83,84 As discussed above, TNF-␣ from macrophages
can augment C5a-induced activation of endothelial cells, as
measured by surface expression of P-selectin. The larger split
products C4b and C3b can bind covalently to cell membranes
and target macrophage adhesion (Figure 2). After being
bound covalently to cell membranes, C3b and its subsequent
cleavage product iC3b serve as accessory adhesion molecules
that strengthen the contact between target cells and macrophages that express CR1 and CR3. A strong correlation
between antibody-mediated complement activation and margination of macrophages in vessels of transplants has been
noted in clinical and experimental specimens.15,16,74,75 In
animal models of transplant vasculopathy, macrophages are
recruited to the arteries rapidly and persist as the neointima
expands.80,85
Antibody and complement have additional stimulatory
effects on macrophages through receptors for Fc and C1q.
Among other functions, macrophage production of C3 and C5
is increased by stimulation through both the Fc44,45 and C1q
receptor.86 This would increase local production of complement by macrophages in vessel walls. Comparisons of atherosclerotic plaques with adjacent normal-appearing artery
have identified local production of many proinflammatory
mediators including mRNA for complement components.47
Both smooth muscle and macrophages appear to be responsible for this response. We have found that local production
of C6 by infiltrating inflammatory cells in the allograft is
increased during rejection.46
Macrophages may be integral to transplant vasculopathy
because macrophages continue to function in spite of current
immunosuppressive treatment. In fact, in the first weeks after
transplantation, when patients receive maximum immunosuppression, macrophages are found routinely in biopsies at sites
of ischemia/reperfusion injury.
Antiinflammatory Effects of Complement
on Macrophages
The interaction of complement split products with macrophages does not inevitably induce mediators of acute inflammation. Deficiencies of C1 and C4 result in decreased
capacities to clear apoptotic cells and increased incidences of
autoimmunity in both humans and mice. Several complement
components have been demonstrated to aid in the noninflammatory clearance of apoptotic cells by macrophages.87 C1q,
MBL, and CRP can initiate this clearance by binding to
phosphatidylserine exposed on the flipped plasma membranes. Phagocytosis induced by C1q, C4b, and iC3b is
accompanied by decreased production of IL-12 and IFN␥.63
C5 activation is the tipping point between noninflammatory
versus inflammatory responses to complement. As discussed
in the previous section, C5a in particular causes macrophages
to modulate Fc and complement receptors toward a heightened activated state.
Localization of B Lymphocytes in Coronary
Arteries With Chronic Vasculopathy
Complement activation by antibodies is a powerful feedback
loop for increased antibody production because C3d, the final
end product of regulation of C3b by factor I, is the ligand for
CR2 (CD21) on B lymphocytes. Fearon and colleagues88
have demonstrated that antigens bearing 2 and 3 copies of
C3d are 1000- to 10 000-fold more immunogenic than antigen alone. This mechanism allows C3d to function as a
molecular adjuvant for antibody production. For this mechanism to occur, the B lymphocyte is best stimulated by both
Wehner et al
Antibody and Complement in Transplant Vasculopathy
199
Figure 4. Heat map for immunoglobulin
cluster in microarray data from human
coronary arteries. Immunoglobulin genes
were generally not or only slightly
upregulated in coronary arteries from 6
hearts with dilated cardiomyopathy
(DCM) and from 6 other hearts with atherosclerosis. In contrast, these genes
were upregulated at least 5-fold in 10 of
12 coronary arteries from cardiac transplants with vasculopathy.
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the antigen and C3d. Conventionally, B lymphocytes and
plasma cells are thought to reside in the spleen and bone
marrow,89,90 but substantial numbers of B cells and plasma
cells have been demonstrated in transplants that have poor
outcomes. To date, most of these observations have been in
renal transplants, and they have not been related to arterial
vasculopathy.91–95
We have found prominent B-cell and plasma cell infiltrates
in coronary arteries with vasculopathy. Using DNA microarrays, we established gene expression profiles of coronary
arteries from 24 human heart explants recovered in the
operating room at the time of transplantation. Coronary
arteries from 3 groups of heart explants were studied: (1)
hearts with dilated cardiomyopathy without coronary lesions
(n⫽6); (2) hearts with native atherosclerosis (n⫽6); and (3)
first cardiac transplants that were replaced because of coronary vasculopathy (n⫽12). The transplants were removed 3
to 14 years (average⫽6.9) after transplantation. Genes for
immunoglobulins (IgG3, ␭, and ␬) as well as CR2 that are
expressed by B lymphocytes were upregulated in coronaries
with vasculopathy compared with native atherosclerosis or no
lesions. Nine probes for ␬ and ␭ light chains were consistently increased in 11 of 12 cardiac allograft vasculopathy
samples. In 5 of these samples, these probes were increased in
the range of 5- to ⬎25-fold (Figure 4).
The localization of B cells and plasma cells was investigated in coronary arteries from 8 of the patients with severe
transplant vasculopathy. Immunohistology demonstrated that
infiltrates of B cells (CD20⫹) and plasma cells (CD138⫹)
were characteristic of coronary arteries with transplant vasculopathy. Linear arrays of these cells often infiltrated along
the elastic layers under the thickened neointima. Larger
numbers of lymphoid cells extended into the adventitia,
where they often formed nodules with distinct inner zones of
B lymphocytes surrounded by T lymphocytes (Figure 5).
Histology from 5 of the 8 patients with severe transplant
vasculopathy showed nodules of compartmentalized B and T
lymphocytes. Plasma cells were often found scattered on the
outside of the nodules. The presence of nodules was not
correlated with the age or sex of the patient, length of graft
survival, or number of HLA mismatches. Stains for ␬ and ␭
light chains and capillary gel electrophoresis of the immunoglobulin heavy chain demonstrated that the plasma cells were
polyclonal, suggesting that the lymphoid nodules were not
caused by posttransplant lymphoproliferative disease. Thaunat et al96 have reported similar findings in coronary arteries
from 5 cardiac transplants.
To exclude the possibility that these nodules were merely
the sequela of open heart surgery, coronary arteries from 6
control patients were examined. These patients had experienced open heart surgery 7 to 10 years before their hearts
were explanted for a first cardiac transplant: 2 patients had
valve replacements, 3 patients had coronary artery bypass
grafts, and 1 patient had a coronary stent. Minimal inflammatory infiltrates and no lymphoid nodules were seen in these
control coronary arteries. The control studies support the
concept that the infiltrates and lymphoid nodules developed
in response to the allogeneic transplant.
The potential role of these lymphoid nodules in antigen
presentation and antibody production has been discussed, but
few data are available. Thaunat et al97 used segmental aortic
grafts in rats to demonstrate that lymphoid nodules formed
adjacent to these large arteries were associated with the
production of antibodies to histocompatibility antigens.
Ectopic lymphoid nodules with distinct B and T lymphocyte zones are frequently formed in tissues affected by
chronic autoimmune diseases, such as thyroiditis, diabetes,
and rheumatoid arthritis. This has been referred to as lymphoid neogenesis. Experimentally, transgenic expression of
certain chemokines has been shown to initiate the recruitment
and organization of lymphocytes into functional appearing
lymphoid structures.98 Ectopic expression of CXCL13 (Blymphocyte chemoattractant), CCL21 (secondary lymphoid
tissue chemokine), and CCL19 (Epstein–Barr virus–induced
chemokine) induce the development of lymph node–like
structures through stimulation of lymphotoxin. All of these
200
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February 2, 2007
Figure 5. Epicardial coronary artery with
vasculopathy 3.5 years after cardiac
transplantation stained by immunoperoxidase for T lymphocytes (CD3; top row),
B lymphocytes (CD20; middle), and
plasma cells (CD138; bottom). Lowpower view (⫻10 original magnification;
left column) demonstrates multiple lymphoid nodules in and adjacent to the
adventitia. Higher-power views (⫻40 and
⫻64 original magnification) of 2 representative nodules (single and double
arrows) illustrate the compartment of the
T lymphocytes surrounding the B lymphocytes. Plasma cells are generally
located outside the nodules.
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structures contain B- and T-cell zones, but those elicited by
CXCL13 and CCL21 are larger and form more rapidly.
CXCL12 (stromal cell– derived factor 1) induces small lymphoid nodules with a preponderance of B lymphocytes and
plasma cells. Steinmetz et al99 described 4 renal biopsies
obtained in the first 4 to 9 days after transplantation that
contained nodules of B cells that stained with CXCL13 and
the corresponding CXCR5 receptor.
In experimental models, lymphoid neogenesis has been
described in 2 models of transplantation.97,100 Lakkis and
colleagues100 described organized nodules of lymphocytes
with T- and B-lymphocyte compartments in heterotopic
mouse hearts undergoing chronic rejection. These experimental heart grafts have a couple of differences with human heart
transplants. First, the heterotopic transplants are located in the
peritoneal cavity. Second, mouse coronary arteries penetrate
into the myocardium closer to their origin. As a result, these
lymphoid nodules were on the epicardium of the transplanted
mouse heart but not adjacent to the coronaries.
Future Investigations
The potential for antibodies and complement to contribute to
the development of transplant vasculopathy needs to be
explored more fully. Our review of the current information
concerning transplant vasculopathy indicates that the interactions of antibody and complement with platelets and macrophages may be central to injury of arterial intima. The
high-flow conditions of the artery only permit high-affinity
interactions to occur. Antibodies and complement have been
demonstrated to bind arterial endothelium and release von
Willebrand factor, which can ensnare platelets and macrophages on the endothelial surface. Complement split products
can pivotally modulate the mediators secreted by macrophages. These interactions need to be investigated more
completely.
Clinically and experimentally, improved methods of testing for antibodies and complement will allow more accurate
assessment of these mediators in transplant recipients with
vasculopathy. In addition, more patients with demonstrable
alloantibodies are being transplanted. In the field of cardiac
transplantation, patients who are supported with left ventricular assist devices have an increased incidence of antibodies
to HLA. Acute rejection caused by antibodies and complement has been treated by combinations of plasmapheresis,
intravenous ␥-globulin and monoclonal antibodies to CD20
on B lymphocytes. The effect of these treatment modalities
on the development of coronary vasculopathy is unknown.
This cohort of patients with documented antibody in their
circulation and complement deposition their transplants will
provide a large database to correlate the effects of antibodies
and complement with the development of vasculopathy.
The increased clinical interest in antibodies and complement has been followed by an increase in experimental
studies. The development of experimental models has been
aided by a variety of animals with useful genetic deficiencies
of selective antibody and complement components, regulators, and receptors. Potent inhibitors for complement fragments and receptors have also been synthesized.
These advances will permit a fuller understanding of the
contributions of different antibodies and complement components to transplant vasculopathy.
Summary
Antibodies and complement can induce vasculopathy in
experimental cardiac transplants in the absence of T or B
lymphocytes. In the clinical setting, it is likely that antibodies
augment platelet and macrophage function through activation
of complement. The initial mechanisms stimulated by antibodies and complement are the expression of chemotaxis and
adhesion molecules by endothelial cells and platelets.
P-selectin promotes interactions among macrophages, plate-
Wehner et al
Antibody and Complement in Transplant Vasculopathy
lets, and endothelial cells. The central role of macrophages is
enhanced by signaling through receptors for Fc and complement split products. Complement split products also modulate
T- and B-lymphocyte immunity. Identification of tertiary
lymphoid structures formed within transplants has raised the
possibility that local immune responses contribute to the
antibody responses in chronic graft rejection. Further experimental animal studies will be needed to define the importance of individual Fc and complement receptors to transplant
vasculopathy.
16.
17.
18.
19.
Acknowledgments
We thank Karen Fox-Talbot for expert help with the
immunohistochemistry preparations.
20.
Sources of Funding
21.
Supported by NIH grants K08HL74945, R01-AI42387,
P01HL070295, and P01HL056091.
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22.
Disclosures
None.
23.
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Antibody and Complement in Transplant Vasculopathy
Jennifer Wehner, Craig N. Morrell, Taylor Reynolds, E. Rene Rodriguez and William M.
Baldwin III
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Circ Res. 2007;100:191-203
doi: 10.1161/01.RES.0000255032.33661.88
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