Antibody-mediated episodes of allograft rejection are increasingly recognized in clinical transplantation. This is due to advances in diagnostic methods and therapeutic interventions. Diagnostically, certain split products of complement, most notably C4d and C3d, have been demonstrated to be more sensitive than immunoglobulin as indicators of antibody-mediated rejection (1–5). In addition, the margination of neutrophils or macrophages in and around capillaries has been found to be a valuable clue to investigate complement deposition (6–8). Therapeutically, the application of intravenous (IV) immunoglobulin (Ig), plasmapheresis, monoclonal antibody to CD-20, and other treatment modalities has expanded the feasibility of transplanting patients who have alloantibodies to their prospective donors (9–13). The incidence of antibody-mediated rejection depends upon many variables, including the stringency of diagnostic criteria, the selectivity of biopsies investigated and the proportion of sensitized patients in the population under study (4, 14). As a result, the incidence of diagnosed antibody-mediated rejections in renal and cardiac transplants ranges from about 2% to 50% (2, 7, 14, 15). In the Banff diagnostic classification, the criteria for diagnosing antibody-mediated rejection include the demonstration of donor-specific antibodies in addition to diffuse, strong staining for C4d in peritubular capillaries (4). Even with these stringent criteria, the diagnosis of antibody-mediated rejection is not always accompanied by functional evidence of graft injury (14). The term subclinical rejection has been proposed to describe C4d deposition in biopsies in the absence of graft dysfunction (16).
In humans, sensitization can result from previous transfusions, pregnancies, or transplants. There have been limited experimental animal models and reagents designed to investigate the effects of antibodies and complement on allograft rejection. Although skin grafts can reliably sensitize rats and mice to subsequent organ transplants (17), small animal models of antibody-mediated rejection following sensitization by transfusions or pregnancies have been uncommon. In fact, the antibody responses elicited by transfusions and pregnancies frequently have been associated with enhanced survival of allografts (18–20). In addition, documentation of complement activation in transplants to rats has been hampered by a lack of clinically relevant reagents. Therefore, we have developed a rabbit polyclonal antibody to rat C4d that permits examination of this marker for antibody-mediated rejection. We have applied this diagnostically relevant marker to transplants between congenic C6-deficient and sufficient rats. The use of C6-deficient rats allowed the verification that antibodies and complement are effectors in this model and not just epiphenomena.
MATERIALS AND METHODS
The derivation of PVG congenic rat strains with a C6 deficiency in our colony at the Johns Hopkins University School of Medicine has been described previously (21). The adult PVG.R8 (RT1.AaBu) and PVG.1U (RT1.AuBu) rats used in these studies are mismatched at major histocompatibility complex (MHC) class I antigens. The MHC phenotype of these congenic rats and their C6 levels in the sera were confirmed by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively.
All animals received humane care in compliance with the “Principles of Laboratory Animal Care” and the “Guide for the Care and Use of Laboratory Animals” prepared and formulated by the Institute of Laboratory Animal resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).
MHC and C6 Analysis
The MHC phenotype of each PVG.R8 and PVG.1U rat was confirmed prior to use by labeling peripheral blood leukocytes (PBL) with MN4 (ATCC, Rockville, MD), an IgG mouse monoclonal antibody to RT1.Aa antigen expressed by PVG.R8 rat lymphocytes and OX3 (ECACC, Port Down, Salisbury, UK), an IgG mouse monoclonal antibody to RT1.Bu antigen expressed by PVG.R8 and PVG.1U rat B lymphocytes. The bound monoclonal antibodies were detected with FITC-conjugated rat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA). After incubating for 30 min at 4oC, the cells were washed twice, then resuspended in PBA containing 1% formalin, and measured by a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
A sandwich ELISA was performed to detect C6 using mouse anti-rat C6 monoclonal antibody 3G11 (a gift from Dr. W. Couser, University of Washington, Seattle, WA) to coat 96-well plates. After blocking the uncoated portions of the wells with PBA (phosphate buffered saline containing 0.2% bovine serum albumin and 0.2% NaN3), PVG rat serum samples were serially diluted and incubated in the wells for one hour. Following three washes with PBS-tween, bound rat C6 was detected with a goat anti-human C6 antibody (Calbiochem, La Jolla, CA), which cross reacts with rat C6, followed by sequential incubation with biotin-conjugated donkey anti-goat IgG antibody (Jackson ImmunoReseach, West Grove, PA), horseradish peroxidase- conjugated streptavidin (Zymed, San Francisco, CA) and the substrate o-phenylenediamine (Sigma Chemical Co., St. Louis, MO). The reaction product was quantitated by measuring the optical density at 450 nm with an Emax Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA).
C6-sufficient and C6-deficient female PVG.1U (RT1.Au) rats were transfused via tail vein with 0.5 ml of heparinized whole blood from allogeneic PVG.R8 (RT1.Aa) donor rats or control isogeneic PVG.1U (RT1.Au) rats. Three weeks after transfusion, all blood recipients received a cardiac allograft from PVG.R8 donors. Under the inhalational anesthesia with Isoflurane (Abbott Laboratories, North Chicago, IL) delivered by a vaporizer (Tech 3, SurgiVet/Anesco, Inc., Waukesha, WI), heterotopic heart transplantation was performed using microsurgical techniques as described previously (21, 22). Briefly, 5 ml of ice-cold cardioplegic solution was injected into the aortic root of the donor heart before removal to metabolically arrest the heart. The donor aorta and pulmonary artery were anastomosed to the recipient infrarenal aorta and inferior vena cava, respectively, in an end-to-side fashion with 8-0 prolene suture (Ethicon, Inc., Somerville, NJ), and then the abdomen was closed in layers. After surgery, all recipients were injected subcutaneously with cyclosporine A (CsA) (Bedford Laboratories, Bedford, OH) at a dose of 5 mg/kg on alternate days until rejection or sacrifice 90 days posttransplantation. Cardiac graft function was evaluated by abdominal palpation daily. Graft rejection was defined as total cessation of contractions.
Blood samples from PVG.1U rats were collected by tail bleeding before transfusion, weekly after transfusion, and weekly after allografting until sacrifice. Blood was allowed to clot for 30 min at 37oC and then one hr at 4oC. After centrifugation at 4oC and 2000 rpm (860g) for 10 min, sera were aspirated and stored at −80oC until use.
IgM, IgG, and IgG Subclass Alloantibody Assay
Alloantibodies were measured by flow cytometry on single cell suspensions of cervical lymph nodes from donor strain rats as described previously (18, 19, 23). Briefly, 50 μl of aliquots containing 1.5×105 lymphocytes were incubated with 50 μl of diluted sera (1:4, 1:16, 1:64, 1:128). The washed cells were reacted with 50 μl of PBA containing a mixture of FITC-conjugated F(ab′)2 fragments of goat anti-rat IgG and phycoerythrin-conjugated F(ab′)2 fragments of goat anti-rat μ-chain of IgM (Jackson ImmunoResearch, West Grove, PA). To analyze the IgG subclasses, the washed cells were reacted with 50 μl of PBA containing FITC-conjugated mouse monoclonal antibodies specific for the Fc region of rat isotypes (RG11/39.4 for IgG1, RG7/1.3 for IgG2a and RG7/11.1 for IgG2b; BD Pharmingen, San Diego, CA) at a concentration 1:100 for 30 min on ice. For IgG2c, a biotinylated mouse anti-rat IgG2c monoclonal antibody (A92-1; BD Pharmingen, San Diego, CA) was used and then reacted with FITC-conjugated streptavidin (Invitrogen, Grand Island, NY). The cells were washed twice and fixed in 250 μl of PBS containing 1% formalin prior to analysis with a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA).
Detection of Rat C4d on Sensitized Lymphocytes
Mononuclear cells were isolated from cervical lymph nodes of PVG.R8 rats and 5×105 cells were incubated with serum that was harvested from PVG.1U rats three weeks after they were transfused with blood from a PVG.R8 donor. After washing, the cells were then incubated for 45 min at 37°C in a Gelatin Veronal Buffer Solution with Mg++ and Ca++ (GVB++) containing 10% serum from a C6-deficient PVG.1U rat (21). The cells were washed, and then incubated with phycoerythrin conjugated mouse monoclonal antibody to rat CD3 (clone G4.18, BD Pharmingen, San Diego, CA) and an affinity purified rabbit antibody to rat C4d (24). A secondary FITC conjugated donkey antibody to rabbit IgG with minimal cross-reactivity to mouse and rat proteins (Jackson ImmunoResearch Laboratories, West Grove, PA) was used to detect the affinity purified rabbit antibody to rat C4d. After staining, the cells were fixed in 1% formalin solution and analyzed with a flow cytometer (FACscan, Becton Dickinson). C4d deposition was assessed on the CD3+ population using Cell Quest software (Becton Dickinson, Mountain View, CA).
Full cross sections of cardiac grafts obtained at the time of sacrifice were fixed in acidic methanol (60% methanol, 10% acetic acid, 30% water), embedded in paraffin and sectioned at seven microns. Rejection was assessed on sections that were stained with hematoxylin and eosin. Proliferative arterial lesions were evaluated on sections stained with Verhoeff-van Gieson for elastic tissue. C4d deposition was localized by immunoperoxidase staining with an affinity purified polyclonal rabbit antibody to rat C4d (24). Macrophages were demonstrated by immunoperoxidase staining for CD68 (ED-1; Serotec Inc., Raleigh, NC).
Cardiac Allograft Rejection by C6-sufficient and -deficient Recipients Pretreated with Allogeneic or Isogeneic Blood
Control C6-sufficient PVG.1U rats that were transfused with isogeneic blood rejected their cardiac allografts by six to seven days after transplantation (n=6). Presensitization with an allogeneic blood transfusion caused accelerated rejection of cardiac allografts by C6-sufficient PVG.1U recipients. All five cardiac allografts to presensitized C6-sufficient PVG.1U recipients were rejected four days after transplantation (Table 1). This demonstrated that the sensitization caused by a single allogeneic blood transfusion was sufficient to accelerate rejection of cardiac allografts in this strain combination.
Cardiac allografts to unsensitized C6-deficient PVG.1U rats that were transfused with isogeneic blood continued to function until sacrifice at 90 days (n=6). Sensitization with an allogeneic blood transfusion did not result in accelerated acute rejection in the absence of C6. Instead, four of five cardiac transplants to sensitized C6-deficient recipients maintained variable levels of function until sacrifice at 90 days (Table 1). These results indicated that both the complete acute rejection in unsensitized recipients and the complete accelerated acute rejection induced by sensitization with an allogeneic blood transfusion required the terminal complement components.
Alloantibody Responses to Donor Class I MHC Antigen Elicited by a Single Allogeneic Blood Transfusion in C6-Sufficient and -Deficient Recipients
In both C6-sufficient and -deficient PVG.1U recipients, a single allogeneic blood transfusion from an MHC class I incompatible PVG.R8 donor elicited a transient IgM alloantibody response to RT1.Aa antigens that peaked in the circulation one week after the transfusion and returned to baseline two weeks later (Fig. 1A). The IgG alloantibody response was more gradual and persistent: IgG titers were greater than 64 by one week after transfusion in both C6-sufficient and -deficient PVG.1U recipients and continued to increase over the three weeks before transplantation. The subsequent antigenic challenge with a PVG.R8 cardiac allograft was accompanied by little change in the level of IgM alloantibodies and a progressive increase IgG antibodies in both sensitized C6-sufficient and -deficient recipients (Fig. 1B).
In unsensitized C6-sufficient and -deficient recipients, a PVG.R8 cardiac allograft elicited IgM and IgG alloantibodies with titers greater than 64 by one week after transplantation. Thus, although the recipients that were sensitized with a blood transfusion had higher levels of IgG alloantibodies early after transplantation, the unsensitized recipients produced strong IgM and IgG alloantibodies by one week after transplantation when the C6-sufficient recipients rejected their transplants. In the unsensitized C6-deficient cardiac allograft recipients, the IgG alloantibody response continued to increase until it reached the same levels as the IgG response in sensitized C6-deficient recipients one month after transplantation (Fig. 1B). The IgG alloantibodies remained at this high level until the time of sacrifice at three months (data not shown).
The IgG alloantibodies to RT1.A that were elicited by transfusion were primarily IgG1 and IgG2b subclasses at the time of transplantation (Fig. 2). Donor specific IgG1 and IgG2b alloantibodies remained the primary response after transplantation in both the C6-sufficient and -deficient recipients.
Conventional measurements of alloantibodies by flow cytometry demonstrate antibody binding to donor specific antigens, but they do not demonstrate the capacity to activate and deposit complement. Therefore, the capacity of the alloantibodies to activate complement was confirmed in a flow cytometry assay designed to detect deposition of C4d on cells. This was critical because the dominant IgG subclasses in the alloantibody response have different capacities to activate complement. Sera sampled from the sensitized recipients at the time of transplantation were found to cause deposition of C4d on PVG.R8 cells. The amount of C4d deposited on the PVG.R8 cells correlated with the level of IgG2b and IgG1 in the serum of individual animals (Fig. 3). Sera from recipients of isogeneic transfusions that contained no demonstrable alloantibody at the time of transplantation did not cause C4d deposition on PVG.R8 cells.
Acute C4d Deposition in Hearts Transplanted to Unsensitized and Sensitized Recipients
Donor-specific alloantibody responses were associated with deposition of C4d on the arteries, capillaries, and veins of the cardiac allografts. Accelerated graft rejection by sensitized C6-sufficient recipients was accompanied by an intense staining for C4d on the injured vascular endothelium at the time of rejection four days after transplantation (Fig. 4A). Control unsensitized C6-sufficient recipients that were sacrificed four days after transplantation (n=2) had lower levels of alloantibodies in their circulation. However, moderate diffuse C4d deposits were already detectable in their transplants. Alloantibodies increased in the circulation of control, unsensitized recipients from day four to day seven after transplantation, and C4d deposits in their transplants were stronger and more diffuse by the time of rejection at seven days (Fig. 4B).
Although the C6 deficiency prevented complete acute rejection, alloantibody production was associated with C4d deposition on the endothelium of arteries, capillaries and veins (Fig. 4C and D). Strong, diffuse deposits of C4d were present on the vessels of transplants in sensitized C6-deficent recipients that were sacrificed four days after transplantation (n=3) and in unsensitized C6-deficent recipients that were sacrificed seven days after transplantation (n=3). The demonstration of C4d deposits indicated that the alloantibodies elicited by transplants bound to the target antigens and activated the early components of the complement cascade in the C6-deficient as well as C6-sufficient allograft recipients.
Infiltrates of Neutrophils, Macrophages, and T cells in Accelerated and Acute Rejection of Cardiac Allografts
The composition and intensity of the infiltrates in cardiac transplants were correlated with the rate of rejection. Accelerated acute rejection in the sensitized C6-sufficient recipients was characterized by the presence of neutrophils. Neutrophils were marginated along the C4d-coated vessels and prominent in the interstitial infiltrates at the time of rejection (Fig. 4A). Cardiac transplants in sensitized C6- deficient recipients that were sacrificed at four days contained only low numbers of neutrophils. These neutrophils were also marginated in C4d-coated capillaries of the transplant.
Acute rejection of cardiac allografts in control, unsensitized C6-sufficient recipients was characterized by dense mononuclear cell infiltrates that surrounded arteries as well as pervaded the myocardium. Strikingly distinct localization patterns were found for macrophages and T lymphocytes in these transplants. Immunohistological stains for CD68 demonstrated that large numbers of macrophages were distributed in the capillaries throughout the myocardium (Fig. 4E). Few macrophages were located periarterially. An inverse distribution of T cells was demonstrated by stains for CD8 in these MHC class I incompatible cardiac transplants. Most of the periarterial cells were CD8+ T cells (Fig. 4G). These T cells extended into the lymphatics. Unlike the macrophage infiltrate, relatively few T cells were present in the capillaries.
Significant macrophage and T-cell infiltrates were also present in transplants to C6-deficient recipients. Again in these transplants, T cells were concentrated in periarterial compartments (Fig. 4H) and macrophages were distributed more diffusely in capillaries (Fig. 4F). Thus, there was a significant cellular response to transplants in C6-deficient recipients.
Chronic Changes in Cardiac Allografts to Sensitized C6-deficient Recipients
Sensitization to a blood transfusion exposed the subsequent cardiac allograft to increased IgG alloantibodies from the time of transplantation through the first month (Fig. 1B). After that time the IgG alloantibody levels were high in both the sensitized and unsensitized cardiac allograft recipients.
Although some transplant function was maintained for over 40 days in the five sensitized C6-deficient recipients, these grafts had significantly more pathology than their unsensitized counterparts. One graft was completely rejected 41 days after transplantation to a sensitized recipient, and three of the four grafts that survived 90 days in sensitized recipients had extensive areas of interstitial fibrosis. Notably, more than a quarter of the large arteries developed accelerated graft arteriosclerosis (AGA) in the sensitized recipients (Fig. 5). These lesions were characterized by α-actin positive smooth muscle cells forming a neointima inside the internal elastic lamina and luminal compromise. In contrast, accelerated graft arteriosclerosis was found in less than 20% of the larger arteries in five of six grafts from unsensitized C6-deficient recipients (Fig. 5).
Sensitization by blood transfusion is a complex process. In both humans and rats, prior transfusions even from the specific donor can induce either sensitization or improved graft survival (19, 25). In humans, transfusions can be a cause of sensitization that either prevents transplantation or requires intensive treatment with modalities such as plasmapheresis and IVIg. Even with these treatments, antibody-mediated rejections episodes are encountered (10).
In rats, the effects of blood transfusions are dependent on the donor and recipient strains studied. Prolonged graft survival occurs in strain combinations that blood transfusions fail to elicit a switch from an IgM to an IgG alloantibody response (18–20). In contrast, increased graft injury results in strain combinations that blood transfusions elicit a strong IgG alloantibody response (18, 26, 27). Using DA rats as donors, Yang et al. (27) demonstrated that transfusion-induced antibodies to MHC class I antigens, but not antibodies to MHC class II antigens, were able to cause rejection of allografts in RT1u nude recipients.
In our previous studies with C6-deficient rats, we have investigated the effects membrane attack complex (MAC) on antibody-mediated rejection. A spontaneous genetic C6 deficiency was discovered in the PVG rat strain (17, 28–30). Stahl and co-workers (31) identified a 31 base pair deletion in exon 10 of the C6 gene. The absence of C6 terminates the complement cascade after C5 cleavage and prevents the assembly of MAC. As a result, serum from C6-deficient rats cannot lyse cells even in the optimal conditions of in vitro hemolytic assays (28, 30). Although the C6 deficiency does not alter the ability of these animals to reject skin allografts (17, 32), it does prolong cardiac allograft survival by several days or weeks depending upon the strain combination (17, 21, 22, 32, 33). This difference in survival does not reflect an absence of a cellular response to cardiac allografts. Indeed, as illustrated in the present experiments, there is a vigorous infiltrate of T cells in cardiac allografts to C6-deficient recipients that would require treatment in a clinical transplant. The experimental grafts continue to function in the C6-deficient recipients because heterotopic cardiac allografts, unlike orthotopic cardiac allografts, have only a minimal functional demand. Therefore, heterotopic allografts can tolerate severe rejection and continue contract as long as the vascular support is not destroyed.
In the present studies, we have examined the involvement of MAC in rejection of cardiac allografts by sensitized recipients. A single allogeneic transfusion of blood prior to transplantation elicited IgM and IgG responses that were equivalent in C6-sufficient and -deficient recipients. Transplants in both C6-sufficient and -deficient recipients had intense diffuse deposits of C4d on vascular endothelium of arteries, capillaries and veins. However, accelerated acute rejection occurred in the presence of C6 but not in the absence of C6. These data indicate that antibodies require activation of the complement cascade through MAC to cause accelerated acute rejection.
The activation of C1 through C5 is not without consequence because sensitization of C6-deficient recipients caused an accelerated accumulation of macrophages in the capillaries and accelerated graft arteriosclerosis in the arteries. The accumulation of macrophages in capillaries has been emphasized as a diagnostically significant feature of antibody- mediated rejection in human cardiac transplants (7, 34). Macrophages have also been described as a component of antibody-mediated rejection in human renal transplants (8). This clinical observation is consistent with the fact that macrophages have multiple receptors for many biologically active split products of the early complement components. These include receptors for C3a and C5a that can promote chemoattraction. In addition, macrophages have receptors for C4b, C3b and iC3b that can serve to localize macrophages in the transplant (35). This interaction between macrophages and complement is consistent with the colocalization of macrophages and C4d deposits in our experiments.
The localization of T cells was strikingly different that that of macrophages. Whereas macrophage localization correlated with C4d deposition, CD8 T cells localized in these MHC class I incompatible transplants to the periarterial lymphatics where C4d deposits were not detected. T cells do express receptors for C3a and C5a and migrate in response to sites of complement activation (36, 37). This suggests that complement activation may induce increased numbers of T cells and macrophages to exit from the circulation, but that greater numbers of macrophages are retained at the sites of complement activation, while the T cells continue to migrate through to the lymphatics. This distribution of macrophages and T cells would explain the diagnostic finding of macrophages in cardiac transplants during antibody-mediated rejection because endomyocardial biopsies sample primarily capillaries and some small arterioles, but not larger arteries. Therefore, T cells may be missed as the result of sampling limitations if they have accumulated around the larger arteries as in our rat model.
In addition to responding to complement split products, macrophages can also contribute to the complement available locally because they can produce all of the complement components. We have demonstrated that functional C6 produced locally by infiltrating macrophages can be a significant factor in tissue injury and ultimately allograft rejection (22).
Although there was more pathology 90 days after transplantation in C6-deficient recipients that were presensitized with a blood transfusion, the circulating alloantibody response was only increased in the first month after transplantation compared to control cardiac allograft recipients. The cardiac allograft elicited a strong alloantibody response and there was no difference in circulating IgG alloantibody levels between the unsensitized and sensitized recipients by the second and third months after transplantation. Therefore, the finding of more chronic vascular changes in the cardiac allografts in sensitized recipients suggests that the arteries may be vulnerable to injury just after transplantation. This association of chronic vascular injury with early acute rejections has been noted in some clinical studies.
The alloantibodies elicited by a blood transfusion were primarily of the IgG1 and IgG2b subclasses. IgG1 antibodies to RT1 generally do not activate complement in vitro, but IgG1 antibodies can augment complement activation by IgG2b antibodies to RT1 (38). The contributions of different subclasses of IgG alloantibody to antibody-mediated rejection are not completely understood. In vitro experiments have demonstrated that antibodies to MHC antigens can have direct effects on endothelial cells in the absence of complement (39, 40). Passive transfer experiments of monoclonal antibodies to MHC antigens indicate that antibodies capable of activating complement greatly augment the effects of noncomplement activating antibodies (40). In the present experiments, in vitro assays of serum samples with high levels of IgG1 and IgG2b alloantibodies caused C4d deposition on allogeneic cells. Therefore, it is likely that both the IgG1 and IgG2b contributed to the C4d deposited in the transplants.
In addition to antibodies, ischemia-reperfusion has been demonstrated to result in activation of complement. Most relevant to our experiments, Sacks and colleagues have reported that ischemia-reperfusion injury in mice is inhibited to a greater extent by a C6 deficiency than by a C4, C3 or C5 deficiency (41). Studies on complement activation by ischemia-reperfusion in rats usually require 30 min or more of warm ischemia (42, 43). To limit the effects of ischemia in our experiments, warm ischemia was minimized during transplantation by the injection of ice-cold cardioplegic solution into the aortic root of the donor heart to metabolically arrest the heart before removal. As a result, we have found minimal C4d deposits in control cardiac allografts harvested two days after transplantation.
In summary, presensitization by allogeneic blood transfusions accelerated graft rejection in the presence of the complete complement cascade in this high responder rat combination. When the complement cascade is terminated at C6, presensitization results in increased deposition of split products of early complement components together with macrophage infiltrates and increased vasculopathy.
This work was supported by National Institutes of Health grants R01-HL63948, R01-AI42387, and P01-HL56091.
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