Kazunori Murata

Mechanisms of complement activation, C4d deposition, and their contribution to the pathogenesis of antibody-mediated rejection

Complement split products have emerged as useful markers of antibody-mediated rejection in solid organ transplants. One split product, C4d, is now widely accepted as a marker for antibody-mediated rejection in renal and cardiac allografts. This review summarizes the rationale for the use of C4d as a marker of antibody-mediated rejection, along with the clinical evidence supporting its use in the clinical diagnosis of antibody-mediated rejection. Antibody-independent mechanisms by which C4d can be activated by the classical and lectin pathways of complement activation are also identified. Finally, mechanisms by which complement activation stimulates effector cells (neutrophils, monocytes, macrophages, platelets, and B and T lymphocytes) as well as target cells (endothelial cells) are discussed in relation to antibody-mediated allograft rejection.

Synergistic deposition of C4d by complement-activating and non-activating antibodies in cardiac transplants

The role of non‐complement‐activating alloantibodies in humoral graft rejection is unclear. We hypothesized that the non‐complement‐activating alloantibodies synergistically activate complement in combination with complement‐activating antibodies. B10.A hearts were transplanted into immunoglobulin knock out (Ig‐KO) mice reconstituted with monoclonal antibodies to MHC class I antigens. In allografts of unreconstituted Ig‐KO recipients, no C4d was detected. Similarly, reconstitution with IgG1 or low dose IgG2b alloantibodies did not induce C4d deposition. However, mice administered with a low dose of IgG2b combined with IgG1 had heavy linear deposits of C4d on vascular endothelium. C4d deposits correlated with decreased graft survival. To replicate this synergy in vitro, mononuclear cells from B10.A mice were incubated with antibodies to MHC class I antigens followed by incubation in normal mouse serum. Flow cytometry revealed that both IgG2a and IgG2b synergized with IgG1 to deposit C4d. This synergy was significantly decreased in mouse serum deficient in mannose binding lectin (MBL) and in serum deficient in C1q. Reconstitution of MBL‐A/C knock out (MBL‐KO) serum with C1q‐knock out (C1q‐KO) serum reestablished the synergistic activity. This suggests a novel role for non‐complement‐activating alloantibodies and MBL in humoral rejection.

In Vivo Platelet–Endothelial Cell Interactions in Response to Major Histocompatibility Complex Alloantibody

Platelets recruit leukocytes and mediate interactions between leukocytes and endothelial cells. Most studies examining this important platelet immune function have focused on the development of atherosclerosis, but similar mechanisms may contribute to acute and chronic vascular lesions in transplants. Platelets have been described as markers of transplant rejection, but little investigation has critically examined a role for platelets in transplant vasculopathy and, in particular, alloantibody-mediated transplant rejection. We now demonstrate using a skin transplant model that alloantibody indirectly induces platelet activation and rolling in vivo. Repeated IgG2a alloantibody injections result in sustained platelet–endothelial interactions and vascular pathology, including von Willebrand factor release, small platelet thrombi, and complement deposition. Maintenance of continued platelet–endothelial interactions are dependent on complement activation. Furthermore, we demonstrate that platelets recruit leukocytes to sites of alloantibody deposition and sustain leukocyte–endothelial cell interactions in vivo. Taken together, our model demonstrates an important role for platelets in alloantibody induced transplant rejection.

C4d Deposition and Clearance in Cardiac Transplants Correlates With Alloantibody Levels and Rejection in Rats

Antibody‐mediated rejection of human cardiac transplants is correlated with C4d deposits and macrophage infiltrates in capillaries of endomyocardial biopsies. We produced an antibody to rat C4d to study C4d deposition and clearance in Lewis rats that were sensitized with a blood transfusion from DA rats 7, 14 or 21 days before cardiac transplantation. Cyclosporin A (CsA) immunosuppression was initiated after transplantation at a dose that inhibited graft rejection, antibody production and C4d deposition in unsensitized recipients. Blood transfusion elicited high levels of circulating IgG alloantibodies, predominantly of the complement‐activating IgG2b subclass, that peaked 14 days after transplantation. At this time, macrophages accumulated in capillaries, and C4d deposits were diffuse and intense on arteries, capillaries and veins. Grafts that survived 90 days in sensitized recipients still had deposits of C4d that were associated with increased interstitial fibrosis and vasculopathy in arteries. Clearance of C4d was determined by retransplanting DA cardiac allografts from Lewis recipients back to DA recipients. C4d deposits were decreased to minimal levels within 5 days after retransplantation. Thus, C4d deposition is not limited to the capillaries, but extends throughout the arterial tree, and despite formation of a covalent bond, C4d is cleared within days.

The involvement of FcR mechanisms in antibody-mediated rejection.

Background. Antibody-mediated rejection is characterized by macrophage margination against vascular endothelium. The potential interactions triggered by antibodies between endothelial cells (EC) and macrophages have not been examined thoroughly in transplants. We used in vivo and in vitro models of antibody-mediated rejection. Methods. Passive transfer of monoclonal alloantibodies (Allo-mAbs) to donor major histocompatibility complex-class I antigens was used to restore acute rejection of B10.A (H-2a) hearts to C57BL/6 (H-2b) immunoglobulin knockout (IgKO) recipients. Intragraft cytokine mRNA expression was measured by real-time polymerase chain reaction. In vitro, mouse EC were cultured in the presence of Allo-mAbs to donor major histocompatibility complex class I antigens and mononuclear cells. Levels of cytokines in culture supernatants were determined in enzyme-linked immunosorbent assay. Results. Expression of MCP-1, IL-6 and IL-1&agr; mRNA was higher in rejecting transplants from recipients treated with Allo-mAbs compared to non-rejecting transplants. EC sensitized with Allo-mAbs produced high levels of MCP-1 and KC. The addition of macrophages to sensitized EC stimulated high levels of IL-6 in addition to MCP-1, KC, Rantes, and TIMP-1. The levels of MCP-1 and IL-6 were significantly lower in co-cultures of EC sensitized with IgG1 Allo-mAbs in the presence of mononuclear cells from Fc&ggr;-Receptor III KO (Fc&ggr;RIII-KO) graft recipients compared to co-cultures with wild-type cells. The levels of both cytokines were also lower in co-cultures of EC stimulated with F(ab′)2 fragments of antibody. Conclusions. Our findings indicate that IgG1 Allo-mAbs to major histocompatibility complex class I antigens can augment graft injury by stimulating EC to produce MCP-1 and by activating mononuclear cells through their Fc receptors.

Antibody and complement mediated injury in transplants following sensitization by allogeneic blood transfusion

Background. Many patients on the waiting list for transplants are sensitized from previous blood transfusions, pregnancy, or transplants. We investigated the role of complement in acute and chronic pathology in hearts transplanted to sensitized rats. Methods. Blood was transfused from allogeneic PVG.R8 rats or control isogeneic PVG.1U rats to C6-sufficient and -deficient PVG.1U rats. Three weeks later hearts were transplanted from PVG.R8 donors and low-dose cyclosporin A was initiated. Results. Allogeneic but not isogeneic blood transfusion elicited strong immunoglobulin (Ig) M, IgG1 and IgG2b alloantibody responses. Sensitization caused accelerated acute rejection of cardiac allografts by C6-sufficient recipients (4 days). In contrast, allografts functioned over 40 days in all C6-deficient recipients, but sensitization caused increased interstitial fibrosis and chronic vasculopathy. Circulating alloantibodies were associated with deposits of C4d on the vascular endothelium together with pericapillary accumulation of neutrophils and macrophages in the grafts. In contrast, T cells accumulated in periarterial lymphatics that did not have C4d deposits. Conclusions. Presensitization by allogeneic blood transfusion causes accelerated acute graft rejection in the presence of the complete complement cascade. In the absence of C6, macrophages colocalized with deposits of C4d and T cells accumulated in the periarterial lymphatics.

Carbon dioxide pneumoperitoneum alters acute-phase response induced by lipopolysaccharide.

Background: As laparoscopic surgery continues to expand in scope, septic patients will be exposed to carbon dioxide (CO 2) pneumoperitoneum in increasing numbers. The biologic advantages or disadvantages of laparoscopic surgery in the setting of sepsis/inflammation are not known. In a rat model, we investigated whether CO 2 pneumoperitoneum alters the inflammatory response induced by bacterial lipopolysaccharide (LPS). Methods: Male rats were injected via the penile vein with LPS (1 mg/kg). Five hours later, the animals (n = 5) were subjected to CO 2 pneumoperitoneum (group I) for 1h; the animals of group II (n = 5) served as controls (no pneumoperitoneum). At 6 h, all animals were killed and the liver harvested for analysis of hepatic acute-phase gene expression. Total RNA was isolated and analyzed by Northern blot hybridization with probes for alpha-2 macroglobulin (A2M) and detected by autoradiography. The film in the linear range of exposure was quantitated using an imaging system. The signal intensity corresponding to A2M mRNA was normalized by the signal corresponding to 28S rRNA detected by staining with methylene blue. Results: The mRNA levels in group II was 6.5 ± 0.9 vs 2.8 ± 0.4 in group I. As compared with rats that received LPS only, those that received a combination of LPS and CO 2 showed a reduction in A2M mRNA levels (57.4%, p = 0.006). Conclusions: These data demonstrate that the presence of CO 2 pneumoperitoneum reduces the inflammatory response established by LPS. This finding challenges the generally accepted notion that smaller incisions alone account for the observed benefits of laparoscopic surgery. It further suggests that CO 2 pneumoperitoneum — aided laparoscopic surgery impedes the inflammatory response and may therefore offer specific benefits over conventional surgery.

C4d Deposition and Cellular Infiltrates as Markers of Acute Rejection in Rat Models of Orthotopic Lung Transplantation

Background. C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. Methods. We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6−) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. Results. The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6+, but not in C6− recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. Conclusions. The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection.

New tools for laparoscopic division of the pancreas: a comparative animal study.

We tested the hypothesis that the pancreas can be safely divided laparoscopically using non-suture devices. Twelve pigs were randomized into 4 groups: 1) laparoscopic distal pancreatectomy (LDP) using an ultrasonic scalpel; 2) LDP using an ultrasonic scalpel with pancreatic stump suture reinforcement; 3) LDP using a 35-mm laparoscopic linear vascular stapler; 4) LDP using a prototype 35-mm radio-frequency laparoscopic linear vascular stapler. There were no serious complications related to distal pancreatectomy. All groups gained weight by postoperative day (POD) 14. Serum amylase, glucose, electrolytes and total bilirubin levels were measured preoperatively and on POD 1, 3, 7, and 14, and peripancreatic peritoneal fluid amylase levels were measured on POD 7 and 14; all remained normal in all groups. Fewer adhesions to the pancreatic stump were found in the ultrasonic scalpel groups as compared with the stapler groups. Ultrasonic dissection may be the superior means of laparoscopic transection of the pancreas.

Use of a cauterizing laparoscopic linear stapler in intestinal anastomosis

The purpose of this study was to assess the usefulness of a cauterizing laparoscopic linear stapler for intestinal anastomosis. In a porcine model, intestinal anastomoses performed with a standard laparoscopic linear stapler, a cauterizing laparoscopic linear stapler (RF stapler), and a two-layer, hand-sewn technique were compared by measuring bursting pressures at 4 and 7 days after surgery. During surgery, the RF stapler provided better hemostasis than the regular stapler for mesenteric transection. At 4 days, one leak occurred in the RF stapler group, and the bursting pressure in the RF stapler group was significantly lower than the bursting pressures in the regular stapler group and the hand-sewn group. In addition, the bursting pressure was significantly greater in the hand-sewn group than in the regular stapler group at 4 days. By 7 days, there were no differences in bursting pressure among the groups. We recommend that the RF stapler not be used for intestinal anastomosis. However, the device may be beneficial for controlling vasculature.