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Background: Alloantibody can contribute significantly to rejection of heart transplants by activation of complement and interactions with a variety of effector cells, including macrophages and monocytes through activating FcγRI, FcγRIII, FcγRIV, the inhibitory FcγRIIB and complement receptors. These receptors link cellular and humoral immunity by bridging the antibody specificity to effector cells. Activating FcγRs are also involved in serum amyloid P component (SAP)-mediated clearance of apoptotic bodies., Methods: B10.A (H-2a) hearts were transplanted into wild-type (WT) or FcγRIII-knockout (KO) C57BL/6 (H-2b) mouse recipients. Levels of alloantibodies and SAP in the circulation were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively. Intragraft cytokine mRNA expression was measured by real-time polymerase chain reaction. Intragraft deposition of C4d, von Willebrand factor, SAP, and activated caspase 3 was visualized by immunochemistry., Results: B10.A hearts in C57BL/6 FcγRIII-KO recipients were rejected acutely within 6 to 8 days compared with 10 to 14 days in WT. The rejection in FcγRIII-KO was accompanied by higher levels of circulating IgM/IgG alloantibodies and SAP than in WT recipients. Histology in FcγRIII-KO cardiac allograft recipients indicated perivascular margination of monocytes and neutrophils, vascular endothelial cell injury, and intense vasculocentric infiltrates with extensive apoptosis. Higher numbers of apoptotic cells, stronger C4d and SAP deposition, and extensive activated caspase 3 were found in areas of dense pockets of apoptotic blebs in FcγRIII-KO., Conclusions: We propose that absence of FcγRIII is associated with the lack of efficient SAP-mediated clearance of apoptotic cells through FcγRs. Apoptotic cells become immunogenic and induce enhanced inflammation, alloantibody production, and complement activation leading to accelerated cardiac allograft rejection.

Background: Mesenchymal stromal cells (MSCs) are multipotent cells with immunoregulatory capacity that are present in most adult organs. We previously demonstrated that co-culture of C57BL/6 kidney-derived MSCs (KSCs) in syngeneic bone marrow-derived dendritic cell (DC) culture induced a DC phenotype (KSC-DC) with reduced major histocompatibility complex (MHC) class II/increased CD80 expression and ability to suppress T-cell responses., Methods: To study their effects on allogeneic DCs, C57BL/6 KSCs were added to incipient BALB/c DC culture, with surface expression of MHC class II/CD80 measured by fluorescence-activated cell sorting. The ability to stimulate T-cell responses was then assessed in an allogeneic mixed leukocyte response. Next, we isolated either BALB/c (donor) or C57BL/6 (recipient) KSC-DCs from co-culture and measured the tempo of rejection after cotransplantation with islet grafts in a mouse model of islet transplantation. Finally, we measured the effects of KSC-DC stimulation on B-cell proliferation and IgM/IgG production in allogeneic cultures., Results: C57BL/6 KSCs induced a BALB/c DC phenotype with significantly decreased MHC class II, increased CD80 expression, and decreased T-cell stimulatory capacity in the mixed leukocyte response (P<0.01 vs. control). Cotransplantation of donor (BALB/c) but not recipient (C57BL/6) KSC-DCs resulted in significant delay of rejection after islet transplantation (P<0.01 vs. control). Finally, stimulation by KSC-DCs resulted in significantly reduced B-cell proliferation and antibody production in allogeneic culture (P<0.01 vs. control)., Conclusions: Our results highlight an important mechanism of MSC-based immunotherapy and its potential for use in clinical transplantation as prevention of rejection and possibly sensitization.

Antibody-mediated rejection has become critical clinically because this form of rejection is usually unresponsive to conventional anti-rejection therapy, and therefore, it has been recognized as a major cause of allograft loss. Our group developed experimental animal models of vascularized organ transplantation to study pathogenesis of antibody- and complement-mediated endothelial cell injury leading to graft rejection. In this review, we discuss mechanisms of antibody-mediated graft rejection resulting from activation of complement by C1q- and MBL (mannose-binding lectin)-dependent pathways and interactions with a variety of effector cells, including macrophages and monocytes through Fcgamma receptors and complement receptors.

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-1alpha 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 Fcgamma-Receptor III KO (FcgammaRIII-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.

In transplantation, activation of complement has largely been equated to antibody-mediated rejection, but complement is also important in recognition of apoptotic and necrotic cells as well as in modifying antigen presentation to T cells and B cells. As a part of the innate immune system, complement is one of the first responses to injury, and it can determine the direction and magnitude of the subsequent responses. Consequently, the effects of complement in allorecognition and graft rejection are increased when organs are procured from cadaver donors because these organs sustain a series of stresses from brain death, prolonged life support, ischemia and finally reperfusion that initiate proinflammatory processes and tissue injury. In addition, these organs are transplanted to patients, who frequently have been sensitized to histocompatibility antigens as the result of transfusions, pregnancies or transplants. Complement activation generates a series of biologically active effector molecules that can modulate graft rejection by directly binding to the graft or by modifying the response of macrophages, T and B cells of the recipient. However, complement is regulated and the process of regulation produces split products that can decrease as well as increase immune responses. Small animal models have been developed to test these variables. The guide for evaluating results from these models remains clinical findings because there are significant differences between the rodent and human complement systems.

Alloantibodies (AlloAbs) are a clinically significant component of the immune response to organ transplants. In our experimental model, B10.A (H-2a) cardiac transplants survived significantly longer in C57BL/6 (H-2b) immunoglobulin knock-out (IgKO) recipients than in their wild-type (WT) counterparts. Passive transfer of a single 50-200-microg dose of complement-activating IgG2b AlloAbs to IgKO recipients reconstituted acute rejection of cardiac allografts. Although passive transfer of a subthreshold dose of 25 microg of IgG2b or a single 100-200-microg dose of non-complement-activating IgG1 AlloAbs did not restore acute rejection to IgKO recipients, a combination of these AlloAbs did cause acute graft rejection. Histologically, rejection was accompanied by augmented release of von Willebrand factor from endothelial cells. IgG1 AlloAbs did not activate complement on their own and did not augment complement activation by IgG2b AlloAbs. However, IgG1 AlloAbs stimulated cultured mouse endothelial cells to produce monocyte chemotactic protein 1 (MCP-1) and neutrophil chemoattractant growth-related oncogene alpha (KC). TNF-alpha augmented IgG1 induced secretion of MCP-1 and KC. These findings indicate that non-complement-activating AlloAbs can augment injury to allografts by complement-activating AlloAbs. Non-complement-activating AlloAbs stimulate endothelial cells to produce chemokines and this effect is augmented in the milieu of proinflammatory cytokines.

Complement is a multifunctional system of receptors and regulators as well as effector molecules. Both the pathogenic and diagnostic power of complement is based on the capacity of the complement system to amplify innate and adaptive immunity. This amplification is accomplished through two strategies: (1) enzymatic reactions in the complement cascade, and (2) stimulation of leukocytes, platelets and parenchymal cells through specific receptors or receptor-independent pore formation. The mechanisms by which complement mediates and modifies nonspecific inflammation, antibody-mediated injury and T-cell responses are of particular significance to the pathogenesis of transplant rejection. Understanding the mechanisms by which complement integrates the interactions of leukocytes, platelets and parenchymal cells offers opportunities to further refine the diagnosis of rejection.

The lung is known to be particularly susceptible to complement-mediated injury. Both C5a and the membrane attack complex (MAC), which is formed by the terminal components of complement (C5b-C9), can cause acute pulmonary distress in nontransplanted lungs. We used C6-deficient rats to investigate whether MAC causes injury to lung allografts. PVG.R8 lungs were transplanted orthotopically to MHC class I-incompatible PVG.1U recipients. Allografts from C6-sufficient (C6(+)) donors to C6(+) recipients were rejected with an intense vascular infiltration and diffuse alveolar hemorrhage 7 days after transplantation (n = 5). Ab and complement (C3d) deposition was accompanied by extensive vascular endothelial injury and intravascular release of von Willebrand factor. In contrast, lung allografts from C6-deficient (C6(-)) donors to C6(-) recipients survived 13-17 days (n = 5). In the absence of C6, perivascular mononuclear infiltrates of ED1(+) macrophages and CD8(+) T lymphocytes were present 7 days after transplantation, but vascular endothelial cells were quiescent, with minimal von Willebrand factor release and no evidence of alveolar hemorrhage or edema. Lung allografts were performed from C6(-) donors to C6(+) recipients (n = 5) and from C6(+) donors to C6(-) recipients (n = 5) to separate the effects of systemic and local C6 production. Lungs transplanted from C6(+) donors to C6(-) recipients had increased alveolar macrophages and capillary injury. C6 production by lung allografts was demonstrated at the mRNA and protein levels. These results demonstrate that MAC causes vascular injury in lung allografts and that the location of injury is dependent on the source of C6.