Section 4 -

The Role of Peritubular Capillary C4d and Leukocytes in Acute Rejection Alex Magil St. Paul's University Hospital Vancouver, BC, Canada

Introduction It has been customary to regard acute rejection as primarily a cell-mediated event characterized by tubulointerstitial and, in some cases, arterial and/or glomerular mononuclear cell infiltration as exemplified by the Banff classification of renal allograft pathology [1]. It has long been recognized that anti-donor antibodies may play a role in acute rejection in some cases but the demonstration of this had been difficult in practice until recently. More than a decade ago Feucht and colleagues demonstrated peritubular capillary (PTC) deposition of the complement split factor C4d in some cases of acute rejection [2]. C4d is generated by the activation of the classical complement pathway by antigen-antibody reaction and binds covalently to tissue elements at the site of activation. Deposition of C4d along PTC has been shown to be closely correlated with circulating donor-specific antibodies in renal allograft recipients experiencing acute rejection and has been suggested as a marker for antibody-mediated rejection (AMR) [3, 4, 5]. This has enabled the pathologist to suggest the diagnosis of AMR in patients whose biopsies show PTC C4d. However, PTC C4d alone is not diagnostic of AMR and additional criteria must be fulfilled for the diagnosis of AMR, especially the detection of circulating anti-donor specific antibodies with evidence of tissue injury [6]. Acute AMR is often resistant to conventional antirejection therapy [3, 7, 8] but may respond to unconventional measures such as plasma exchange [with tacrolimus-mycophenolate rescue], intravenous immunoglobulin infusion, immunoabsorption, and anti-CD20 monoclonal antibody (rituximab) [8, 9] underlining the importance of recognizing acute AMR in allograft biopsies. Criteria for the Diagnosis of AMR The following criteria have been recommended for the diagnosis of AMR [6]: Circulating anti-donor specific antibody PTC C4d deposition At least one of the following tissue changes: ATN-like - Acute tubular injury with minimal inflammation Capillary - Glomerulitis, neutrphils (PMN) and/or monocyte/macrophages (MO) in PTC capillaries with margination and/or thrombosis Arterial – Transmural inflammation or fibrinoid change The findings of PTC C4d deposition and at least one of the tissue changes are termed "suspicious for AMR" if anti-donor antibody has not been demonstrated. It should be noted that AMR may occur with any of the Banff 97 categories of acute cell-mediated rejection. Detection of PTC C4d PTC C4d can be detected in snap-frozen renal tissue by indirect immunofluorescence or immunoperoxidase with a commercially available mouse monoclonal anti-C4d antibody [3, 10] or in fixed paraffin-embedded tissue by immunoperoxidase with a polyclonal anti-C4d antibody [5]. The Seventh Banff Conference issued criteria for C4d positivity in renal allografts for both cryostat frozen sections using monoclonal antibody and paraffin-embedded sections using polyclonal antibody [11]. Widespread circumferential PTC staining for C4d in either the cortex or medulla, excluding necrotic or scarred areas, is considered a positive result. A strong reaction is required in the frozen sections but not in the paraffin-embedded ones where even weak circumferential staining would be accepted as a positive result. Glomerular staining is almost always seen in biopsies with or without PTC staining and has no specific diagnostic significance in the allograft biopsy. The Prognostic Significance of PTC C4d In addition to its importance in the management of acute rejection, PTC C4d deposition in early acute rejection has been demonstrated to be a long-term prognostic factor associated with relatively poor graft outcome [4, 5, 10, 12]. Early studies had shown an association of PTC C4d deposition with vascular rejection suggesting that PTC C4d may not be an independent predictor of outcome [1, 13]. However, subsequent investigations have shown PTC C4d to be a predictor of long-term graft outcome independent of vascular rejection and a wide variety of clinical parameters as [10, 12, 13]. The Significance of Focal PTC C4d Although diffuse (involving >50% of PTC) staining of PTC for C4d has been included as one of the criteria for the diagnosis of AHR in a recent update of the Banff '97 classification of renal allograft rejection [6], the significance of focal PTC C4d deposition is uncertain [14]. In some studies, patients with diffuse or focal PTC C4d deposition have been grouped together [10, 13, 15] while in others (from one group), only patients with diffuse PTC C4d deposition were considered as being C4d positive [3, 4, 8, 9]. Two previous studies that have compared patients with focal PTC C4d to those with diffuse PTC C4d have not noted any significant differences between them with respect to histological biopsy findings [15] and graft survival [2]. However, in a recent report graft loss occurred more frequently in patients with diffuse PTC C4d compared to those with focal C4d [16]. The results of one study that specifically addressed the issue of focal vs. diffuse PTC C4d deposition suggested that the biopsy and clinical course in patients with focal PTC C4d staining are similar to those in subjects with diffuse PTC C4d (17). The Specificity of PTC C4d as a Marker for AMR Although, as mentioned above, the deposition of PTC C4d in renal allografts has been shown to be closely correlated with circulating anti-donor specific antibodies [3, 4, 5], the demonstration of capillary staining for C4d in heart transplants with ischemic injury but no rejection [18] and within myocardial infarcts in native hearts [19] has suggested that C4d capillary deposition may be associated with ischemic injury. An investigation of preoperative renal transplant biopsies with ischemic damage did not demonstrate PTC C4d staining [20]. Finally, it should be mentioned that the lectin pathway of complement activation produces C4d and the results of a recent study have suggested that the lectin pathway of complement activation may be involved in PTC C4d deposition in renal allografts [21]. Neutrophils (PMN) The association of PTC PMN infiltration and AMR was noted by Trpkov and colleagues about 10 years ago [22]. Other groups have subsequently confirmed this association by demonstrating a correlation of PTC and glomerular PMN numbers with PTC C4d deposition [4, 23]. Whether PMN infiltration is an independent prognostic factor is unknown. Monocyte/Macrophages (MO) MO have long been recognized as a component of the inflammatory infiltrate in acute rejection [24, 25, 26, 27] and increased numbers have been associated with relatively poor graft survival (24-26}. A recent investigation demonstrated a significant association of both glomerular and interstitial MO infiltration with PTC C4d deposition [17]. The correlation of PTC C4d and glomerular MO infiltration has been subsequently confirmed by another group [28]. Transplant glomerulitis (TG) characterized by mononuclear cell infiltration of glomeruli is a well-recognized component of the acute allograft rejection reaction and is observed in a proportion of biopsies demonstrating acute rejection [29, 30]. Both T cells and MO have been demonstrated in the glomeruli in TG [23, 30, 31] and recent reports have suggested an association of TG with peritubular capillary (PTC) C4d deposition [15, 23, 32]. In a recent study, the predominant infiltrating endocapillary mononuclear cell in TG with PTC C4d deposition was shown to be the MO whereas in biopsies with no PTC C4d staining the predominant glomerular leukocyte was the T cell [33]. Although older studies had demonstrated an association of increased MO infiltration in acute rejection with reduced graft survival [24, 25, 26], the close correlation of MO with PTC C4d has raised the question of whether MO infiltration in acute rejection is an independent prognostic factor in the face of PTC C4d deposition's demonstrated adverse effect on graft survival. A recent study, which addressed this issue, did show that increased glomerular, but not interstitial, MO infiltration in acute rejection is an independent predictor of worse graft survival [34]. Plasma Cells The interstitial inflammatory reaction in acute rejection frequently contains a few plasma cells. However, plasma cell-rich infiltrates, which are relatively rare in early acute rejection, fairly frequently occur in late acute rejection [35] and have been shown to predict relatively poor graft outcome [35, 36, 37, 38]. Of interest has been the demonstration of an association of increased numbers of interstitial plasma cells in acute rejection with PTC C4d deposition in two studies [37, 38], one of which also showed that this association was significantly more common in patients with concomitant herpes viral (CMV or EBV) infections [38]. Pathogenesis of PTC Injury in AMR It has been shown that in acute rejection anti-endothelial antibodies bind to endothelial cells [39] and both complement and antibodies activate endothelial cells with increased expression of adhesion molecules (ICAM, VCAM, ELAM-1), cytokines (PDGF,bFGF) and chemokines (MCP-1, CCL5 (RANTES), CXCL8 (IL-8), IL-1α, IL-6) [39], [reviewed in 40]. This leads to recruitment and activation of leukocytes which can damage endothelium [40]. In a recent study Liptak et al. demonstrated PTC lysis and increased rate of apoptosis of endothelial cells in renal allografts undergoing AMR [41]. Additional evidence of PTC injury associated with AMR has been the demonstration of a correlation of marked PTC basement membrane lamellation with PTC C4d deposition in chronic rejection [42]. References Racusen LC, Solez K, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713-723, 1999 Feucht HE, Schneeberger H, Hillebrand G, et al: Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int 43:1333-1338, 1993 Collins AB , Schneeberger EE, Pascual MA, et al: Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol 10:2208-2214, 1999 Mauiyyedi S, Crespo M, Collins AB, et al: Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 13:779-787, 2002 Böhmig GA , Exner M, Habicht A, et al: Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol 13:1091-1099, 2002 Racusen LC, Colvin RC, Solez K, et al: Antibody-mediated rejection criteria – an addition to the Banff '97 classification of renal allograft rejection. Am J Transplant 3:708-714, 2003 Halloran PF, Schlaut J, Solez K, Srinivasa NS : The significance of the anti-class I response. II. Clinical and pathologic features of renal transplants with anti-class I-like antibody. Transplantation 53:550-555, 1992 Pascual M, Saidman S, Tolkoff-Rubin N, et al: Plasma exchange and tacrolimus-mycophenolate rescue for acute humoral rejection in kidney transplantation. Transplantation 66:1460-1464, 1998 Böhmig G, Regele H: Diagnosis and treatment of antibody-mediated kidney allograft rejection. Transpl Int 16:773-787, 2003 Herzenberg AM, Gill JS, Djurdjev O, Magil AB : C4d deposition in acute rejection: an independent log-term prognostic factor. J Am Soc Nephrol 13:234-241, 2002 Racusen LC, Halloran PF, Solez K: Banff 2003 meeting report: new diagnostic insights and standards. Am J Transplant 4:1562-1566, 2004 Regele H, Exner M, Watschinger B, et al: Endothelial C4d depositionis associated with inferior kidney allograft outcome independently of cellular rejection. Nephrol Dial Transplant 16:2058-2066, 2001 Kluth-Pepper B, Schneeberger H, Lederer SR, et al: Impact of humoral alloreactivity on the suevival of renal allografts. Transplant Proc 30:1772, 1998 Feucht HE: Complement C4d in graft capillaries – the missing link in the recognition of humoral alloreactivity. Am J Transplant 3:646-652, 2003 Nickeleit V, Zeiler M, Gudat F, et al: Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol 13:242-251, 2002 Poduval RD , Kadambi PV, Josephson MA, et al: Implications of immunohistochemical detection of C4d along peritubular capillaries in late acute renal allograft rejection. Transplantation 79:228-235, 2005 Magil AB , Tinckam KJ: Focal peritubular capillary C4d deposition in acute rejection. Nephrol Dial Transplant 21:1382-1388, 2006 Baldwin WM III, Samaniego-Picota M, Kasper EK, et al: Complement deposition in early cardiac transplant biopsiesis associated with ischemic injury and subsequent rejection episodes. Transplantation 68:894-900, 1999 Yasojima K, Schwab C, McGeer EG, et al: Human heart generates complement proteins that are upregulated and activated after myocardial infarction. Circ Res 83:860-869, 1998 Haas M, Rattner LE, Montgomery RA: C4d staining of perioperative renal transplant biopsies. Transplantation 74:711-717, 2004 Imai N, Nishi S, Alchi B, et al: Immunohistochemical evidence of activated lectin pathway in kidney allografts with peritubular capillary C4d deposition. Nephrol Dial Transplant (in press) Trpkov K, Campbell P, Pazderka F, et al: Pathologic features of acute renal allograft rejection associated with donor-specific antibody: analysis using the Banff grading schema. Transplantation 61:1586-1592, 1996 Magil AB , Tinckam K: Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection. Kidney Int 63:1888-1893, 2003 Hayry P, von Willebrand E: Monitoring of human renal allograft rejection with fine-needle aspiration cytology. Scand J Immunol 13:87-97, 1981 Harry TR, Coles GA, Davies M, et al: The significance of monocytes in glomeruli of human renal transplants. Transplantation 37:70-73, 1984 Om A, Baquero A, Raja R, et al: The prognostic significance of the presence of monocytes in glomeruli of renal transplant allografts. Transplant Proc 19:1618-1622, 1987 Grimm PC, McKenna R, Nickerson P, et al: Clinical rejection is distinguished from subclinical rejection by increased infiltration by a population of activated macrophages. J Am Soc Nephrol 10:1582-1589, 1999 Sund S, Reisaeter AV, Scott H, Mollnes TE, Hovig T: Glomerular monocyte/macrophage influx correlates strongly with complement activation in 1-week protocol kidney allograft biopsies. Clin Nephrol 62:121-130, 2004 Axelsen RA, Seymour AE, Mathew TH, Canny A, Pascoe V. Glomerular transplant rejection: a distinctive pattern of early graft damage. Clin Nephrol 23:1-11, 1985 Hiki Y, Leong AS-Y, Mathew TH, Seymour AE, Pascoe V, Woodroffe AJ. Typing of intraglomerular mononuclear cells with transplant glomerular rejection. Clin Nephrol 26:244-249, 1986 Bishop GA, Hall BM, Duggin GG, Horvath JS, Sheil AG, Tiller DJ. Immunopathology of renal allograft rejection analyzed with monoclonal antibodies to mononuclear cell markers. Kidney Int 29:708-717, 1986 Sund S, Hovig T, Reisaeter AV, Scott H, Bentdal Ø, Mollnes TE. Complement activation in early protocol kidney graft biopsies after living-donor transplantation. Transplantation 75:1204-1213, 2003 Magil AB: Infiltrating cell types in transplant glomerulitis: relationship to peritubular capillary C4d deposition. Am J Kidney Dis 45:1084-1089, 2005 Tinkam KJ, Djudjev O, Magil AB: Glomerular monocytes predict worse outcomes after acute renal allograft rejection independent of C4d status. Kidney Int 68:1866-1874, 2005 Charney DA, Nadasdy T, Lo AW, Racusen LC: Plasma cell-rich acute renal allograft rejection. Transplantation 68:791-797, 1999 Meehan SM, Domer P, Josephson M, et al: The clinical and pathologic implications of plasmacytic infiltrates in percutaneous renal allograft biopsies. Hum Pathol 32:205-215, 2001 Desvaux D, Le Gouvello S, Pastural M, et al: Acute renal allograft rejections with major interstitial oedema and plasma cell-rich infiltrates: high gamma-interferon expression and poor clinical outcome. Nephrol Dial Transplant 19:933-939, 2004 Aiello FB, Calabrese F, Rigotti P, et al: Acute rejection and graft survival in renal transplanted patients with viral diseases. Mod Pathol 17:189-196, 2004 Lucchiari N, Panajotopoulos N, Xu C, et al: Antibodies eluted from acutely rejected renal allografts bid to and activate human endothelial cells. Hum Immunol 61:518-527, 2000 Colvin RB, Smith RN: Antibody-mediated organ-allograft rejection. Nat Rev Immunol 5:807-817, 2005 Liptak P, Kemeny E, Morvay Z,et al: peritubular capillary damage in acute humoral rejection: an ultrastructural study on human renal allografts. Am J Transplant 5:2870-2876, 2005 Regele H, Böhmig G, Habicht A, et al: Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 13:2371-2380, 2002