Departments of Surgery, Immunology and Pediatrics, Mayo Clinic, Rochester, Minnesota.
Correspondence to Dr. Jeffrey L. Platt, Transplantation Biology, 2-66 Medical Sciences Building, Mayo Clinic, Rochester, Minnesota 55905. Phone: 507-538-0313; Fax: 507-284-4957; E-mail: platt.jeffrey{at}mayo.edu
Research over the past ten years has revealed that humoral rejectionof organ transplants is associated with C4d deposits along thecapillaries of the graft (1–3) and, hence, it is thoughtthat C4d provides a valuable diagnostic and prognostic marker.In this issue of JASN, Regele et al. (4) now suggest that chronicrejection might also be monitored by the detection of C4d. Inthis brief commentary, I shall consider what imperatives driveefforts to detect C4d in organ grafts and why and in what wayuse of C4d as a predictor of graft outcome might imperil thegraft and the recipient.
Acute humoral rejection and some forms of chronic rejection(5) are initiated by the binding of anti-donor antibodies tothe endothelial lining of blood vessels in the graft. Antibodybinding triggers assembly of C1qrs complexes that in turn catalyzethe cleavage of complement components C4 and C2, as modeledin Figure 1. C4b, generated in this way, forms amide or esterbonds with nearby proteins or saccharides, such as componentsof the endothelial cell surface and then associates with C2ato form the classical C3 convertase, C4b2a (C3 convertases amplifythe complement cascade by cleaving C3 to yield the alternativepathway C3 convertase). Formation of C3 convertase on the surfaceof endothelial cells amplifies activation of complement andthus helps to contain microorganisms in inflammatory thrombiand to promote healing of damaged tissues (6). However, complementactivation may give rise to cell injury or lysis. Hence theintegrity and function of C3 convertase is subject to multilayeredcontrols. One control on C3 convertase is mediated by factorI in plasma, which, together with membrane co-factor protein(7), cleaves C4b to yield C4d (Figure 1), a catalytically inactivefragment. Another control on complement-mediated injury is providedby changes in cellular metabolism induced by sublytic amountsof the membrane attack complex, which render cells less sensitiveto complement-mediated injury. We shall return to these mattersshortly.
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 identifyinghumoral rejection (patients with humoral rejection may not havedetectable levels of anti-donor antibodies because those antibodiesare absorbed by the graft and tissues with humoral rejectionmay 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 prescribeplasmapheresis, cytotoxic therapy, and even irradiation). Therefore,a reliable way to prove the diagnosis of humoral rejection wouldbe most welcome. Is detection of C4d in graft biopsies an infalliblesign of humoral rejection? Sadly for the pathologist and happilyfor the patient, it is not.
Why C4d may fail as a sign of humoral rejection and why it shouldnot 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 reasonto withhold therapy. Second, the presence of C4d and absenceof deposits of Ig or other components of complement may indicatethat the metabolism of endothelium has been modified to enhancethe clearance of immune complexes. Carney et al. (8), Morganet al. (9), and Kerjaschki et al. (10), showed in distinct systemsthat sublytic amounts of terminal complement complexes enhanceshedding and endocytosis of immunoproteins attached to cellsurfaces. Third, C4d can be found in organ transplants with"accommodation" (Figure 2). We discovered accommodation in thecourse of studying ABO-incompatible renal transplants that seemedto function perfectly well despite the presence of anti-bloodgroup antibodies in the blood of the recipients (11–13).Accommodation is thought to result from an acquired resistanceof the graft to humoral injury, and accumulating evidence suggeststhat it is induced by bound anti-donor antibodies and complement(14,15). Fourth, formation of C4d and interaction with C4d receptorson B cells may promote regulation of B cell responses to autoantigens,and interfering with formation of C4d in experimental animalsmakes autoimmunity worse (16). Thus the presence of C4d maybe a marker of endothelial health and humoral immune regulationand thus may be good for the graft. In that light, one mightpause before depleting the antibodies or modifying the complementreactions 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 casesof chronic rejection, but in what fraction of cases is uncertain.Since anti-donor antibodies are efficiently cleared from circulationby binding to the graft and bound antibodies can be efficientlycleared cell surfaces on which complement is activated, routineserologic and immunopathologic studies might underestimate theincidence of humoral-mediated chronic rejection. Thus, detectionof C4d may prove to be a useful marker, as Regele et al. (4)suggest, because unlike other immunoproteins, it may persiston cell surfaces. However, one should not hasten to assume thatC4d deposits and chronic changes are pathogenetically connected.If C4d marks conditions in which blood vessels in the grafthave acquired resistance to humoral injury and if that resistancehas been effective, then one would expect the graft to survivelonger, perhaps long enough to develop chronic lesions fromsome other cause. Whether C4d indicates humoral-mediated chronicrejection or a protected state might be resolved, however, bycareful study of biopsies from long-surviving grafts. One mightbe keen to know whether deposits of C4d coincide anatomicallywith early chronic changes or whether vessel segments with C4dare relatively spared. In the latter case, one might entertainthe radical question of whether anti-donor antibodies and/oractivation of complement leading to formation of C4d could beused to protect a graft against chronic rejection.
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