C4d and the Fate of Organ Allografts

Research over the past ten years has revealed that humoral rejection of organ transplants is associated with C4d deposits along the capillaries of the graft (1–3) and, hence, it is thought that C4d provides a valuable diagnostic and prognostic marker. In this issue of JASN, Regele et al. (4) now suggest that chronic rejection might also be monitored by the detection of C4d. In this brief commentary, I shall consider what imperatives drive efforts to detect C4d in organ grafts and why and in what way use of C4d as a predictor of graft outcome might imperil the graft and the recipient. Acute humoral rejection and some forms of chronic rejection (5) are initiated by the binding of anti-donor antibodies to the endothelial lining of blood vessels in the graft. Antibody binding triggers assembly of C1qrs complexes that in turn catalyze the cleavage of complement components C4 and C2, as modeled in Figure 1. C4b, generated in this way, forms amide or ester bonds with nearby proteins or saccharides, such as components of the endothelial cell surface and then associates with C2a to form the classical C3 convertase, C4b2a (C3 convertases amplify the complement cascade by cleaving C3 to yield the alternative pathway C3 convertase). Formation of C3 convertase on the surface of endothelial cells amplifies activation of complement and thus helps to contain microorganisms in inflammatory thrombi and to promote healing of damaged tissues (6). However, complement activation may give rise to cell injury or lysis. Hence the integrity and function of C3 convertase is subject to multilayered controls. One control on C3 convertase is mediated by factor I in plasma, which, together with membrane co-factor protein (7), cleaves C4b to yield C4d (Figure 1), a catalytically inactive fragment. Another control on complement-mediated injury is provided by changes in cellular metabolism induced by sublytic amounts of the membrane attack complex, which render cells less sensitive to complement-mediated injury. We shall return to these matters shortly.Figure 1.: Complement activation and formation of C4d. (Top) Binding of complement fixing antibodies to a cell surface recruits C1qrs complexes. C1qrs cleaves and activates C4 and C2. C4b formed in this way may form covalent bonds with the cell surface and associate with C2a to form C4b2a, the classical complement pathway C3 convertase. C4b2a catalyzes cleavage of C3 and C5, amplifying complement activation. (Bottom) C3 convertases are controlled by various mechanisms. One mechanism involves cleavage of C4b by factor I plus MCP or C4-binding proteins as cofactors to yield C4d, which is catalytically inactive. Although C4d is catalytically inert, it can interact with C4d receptors on B cells and follicular dendritic cells. These interactions may help to regulate humoral immune responses.Interest in using C4d stems in part from the challenge of identifying humoral rejection (patients with humoral rejection may not have detectable levels of anti-donor antibodies because those antibodies are absorbed by the graft and tissues with humoral rejection may lack deposits of Ig or complement because of tissue injury [2]) and the profound implications of identifying humoral rejection (a diagnosis of humoral rejection may lead clinicians to prescribe plasmapheresis, cytotoxic therapy, and even irradiation). Therefore, a reliable way to prove the diagnosis of humoral rejection would be most welcome. Is detection of C4d in graft biopsies an infallible sign of humoral rejection? Sadly for the pathologist and happily for the patient, it is not. Why C4d may fail as a sign of humoral rejection and why it should not per se prompt therapeutic intervention is instructive. First, when grafts are severely afflicted with vascular rejection, C4d may be absent; thus the absence of C4d should not be reason to withhold therapy. Second, the presence of C4d and absence of deposits of Ig or other components of complement may indicate that the metabolism of endothelium has been modified to enhance the clearance of immune complexes. Carney et al. (8), Morgan et al. (9), and Kerjaschki et al. (10), showed in distinct systems that sublytic amounts of terminal complement complexes enhance shedding and endocytosis of immunoproteins attached to cell surfaces. Third, C4d can be found in organ transplants with “accommodation” (Figure 2). We discovered accommodation in the course of studying ABO-incompatible renal transplants that seemed to function perfectly well despite the presence of anti-blood group antibodies in the blood of the recipients (11–13). Accommodation is thought to result from an acquired resistance of the graft to humoral injury, and accumulating evidence suggests that it is induced by bound anti-donor antibodies and complement (14,15). Fourth, formation of C4d and interaction with C4d receptors on B cells may promote regulation of B cell responses to autoantigens, and interfering with formation of C4d in experimental animals makes autoimmunity worse (16). Thus the presence of C4d may be a marker of endothelial health and humoral immune regulation and thus may be good for the graft. In that light, one might pause before depleting the antibodies or modifying the complement reactions that generate C4d.Figure 2.: C4d deposits in accommodation. Organs transplanted across a humoral immune barrier are not invariably rejected. Under some conditions, the organs can acquire resistance to injury leading to long-term function and survival in the face of anti-donor antibodies. This condition, called accommodation, typically occurs in ABO-incompatible kidney transplants. As shown in this figure, ABO-incompatible transplants with accommodation often have C4d deposits on glomerular (inset) and peritubular capillary endothelium.What about chronic rejection and the findings of Regele et al. (4)? Anti-donor antibodies are thought to underlie some cases of chronic rejection, but in what fraction of cases is uncertain. Since anti-donor antibodies are efficiently cleared from circulation by binding to the graft and bound antibodies can be efficiently cleared cell surfaces on which complement is activated, routine serologic and immunopathologic studies might underestimate the incidence of humoral-mediated chronic rejection. Thus, detection of C4d may prove to be a useful marker, as Regele et al. (4) suggest, because unlike other immunoproteins, it may persist on cell surfaces. However, one should not hasten to assume that C4d deposits and chronic changes are pathogenetically connected. If C4d marks conditions in which blood vessels in the graft have acquired resistance to humoral injury and if that resistance has been effective, then one would expect the graft to survive longer, perhaps long enough to develop chronic lesions from some other cause. Whether C4d indicates humoral-mediated chronic rejection or a protected state might be resolved, however, by careful study of biopsies from long-surviving grafts. One might be keen to know whether deposits of C4d coincide anatomically with early chronic changes or whether vessel segments with C4d are relatively spared. In the latter case, one might entertain the radical question of whether anti-donor antibodies and/or activation of complement leading to formation of C4d could be used to protect a graft against chronic rejection.