J Am Soc Nephrol 10: 2208–2214, 1999 Complement Activation in Acute Humoral Renal Allograft Rejection: Diagnostic Significance of C4d Deposits in Peritubular Capillaries A. BERNARD COLLINS, EVELINE E. SCHNEEBERGER, MANUEL A. PASCUAL, SUSAN L. SAIDMAN, WINFRED W. WILLIAMS, NINA TOLKOFF-RUBIN, A. BENEDICT COSIMI, and ROBERT B. COLVIN Departments of Pathology, Medicine, and Surgery and the Immunopathology and Transplantation Units, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. Abstract. The distinction between acute humoral rejection biopsies (16 of 16). IgM and/or C3 were also present in 19 and (AHR) and acute cellular rejection (ACR) in renal allografts is 44%, respectively. With two-color immunofluorescence, C4d therapeutically important, but pathologically difficult. Since was localized in basement membranes (type IV collagen1) and AHR is probably mediated by antibodies to the donor endo- in the endothelium (Ulex europaeus agglutinin-I1). In ACR, thelium that activate the classical complement pathway, it was no more than trace C4d was found in peritubular capillaries hypothesized that peritubular capillary C4d deposition might (P , 0.0001 versus AHR), and no patient had anti-donor HLA distinguish this group. Renal biopsies (n 5 16) from 10 pa- antibodies (0 of 8); 27% had neutrophils in peritubular capil- tients with AHR who had acute graft dysfunction, neutrophils laries. One of six biopsies with cyclosporin A toxicity had in peritubular capillaries, and a concurrent positive cross- similar C4d deposits, and circulating anti-donor class I anti- match were stained for C4d by immunofluorescence. Control body was detected. Grafts with AHR were lost (40%) more biopsies for comparison showed ACR (n 5 14), cyclosporin A often than those with ACR (0%; P , 0.02). C4d in peritubular toxicity (n 5 6), or no abnormality (n 5 4). Peribiopsy sera capillary walls distinguishes AHR from ACR, is more specific were tested for anti-donor HLA antibody. C4d deposited prom- and sensitive than traditional criteria, and is a potentially inently and diffusely in the peritubular capillaries in all AHR valuable adjunct in the diagnosis of graft dysfunction. Acute humoral rejection (AHR) is a form of renal allograft 9). Unfortunately, immunofluorescence for IgG, IgM, C3, or rejection that typically occurs a few weeks after transplantation fibrin is not as helpful as one might expect, revealing no and is associated with circulating antibodies to donor HLA statistically significant difference between AHR and ACR (8). class I (1), class II (2,3), or non-MHC (4–6) antigens on At present, the diagnosis of AHR is made by identification of endothelium. AHR may coexist with T cell-mediated injury suspicious morphologic features on biopsy with subsequent (acute cellular rejection [ACR]), and probably contributes to serologic confirmation of circulating anti-donor antibodies. graft injury more often than is generally appreciated. Indeed, The prognosis of AHR is uniformly worse than ACR (1,5– cytotoxic anti-donor class I antibody was found in the circu- 8). However, those that recover from the acute episode of AHR lation at the time of acute rejection in 20 to 25% of patients have a similar long-term outcome (8), suggesting that the (1,7). Such antibodies correlate with an increased risk of clin- pathogenetic humoral response can be transient if treated ef- ically severe acute rejection and graft loss (1,7). fectively. Indeed, plasmapheresis combined with mycopheno- Identification of AHR in biopsy specimens has been prob- late mofetil and tacrolimus can be successful in reversing AHR lematic, because no morphologic feature described to date is and restoring graft function (10). These important therapeutic either pathognomonic or universal. Several are helpful in rais- implications of AHR have stimulated our efforts to develop ing the possibility, however, including neutrophils in peritubu- more sensitive and specific diagnostic criteria. lar capillaries, fibrinoid necrosis, and glomerular thrombi (1,7– Materials and Methods Patients Received March 11, 1999. Accepted April 7, 1999. The study group consists of all renal biopsies evaluated from This work was presented in part at the 31st annual meeting of the American Society of Nephrology, October 25, 1998, and has been published in abstract January 1994 to August 1998 (16 biopsies) at the Massachusetts form. General Hospital that met the following inclusion criteria: (1) acute Correspondence to Dr. Robert B. Colvin, Department of Pathology, Massa- graft dysfunction; (2) prominent neutrophils in cortical peritubular chusetts General Hospital, 32 Fruit Street, Boston, MA 02114. Phone: 617- capillaries or fibrinoid necrosis of arteries; and (3) a positive anti- 726-2966; Fax: 617-726-7533; E-mail: [email protected] donor T and/or B cell cross-match in a concurrent serum. These 1046-6673/1010-2208 criteria are generally accepted to favor AHR (11). All but one had a Journal of the American Society of Nephrology negative pretransplant cross-match (patient 4 was weakly positive, Copyright © 1999 by the American Society of Nephrology retrospectively). All except two received a cadaveric kidney; two were J Am Soc Nephrol 10: 2208–2214, 1999 C4d Deposits in Peritubular Capillaries 2209 living related donors (daughter, mother) (Table 1). Maintenance im- patient sera with immunomagnetic beads coated with anti-mouse IgG munosuppression included cyclosporin A (CsA), prednisone, and aza- (15). Flow cytometric cross-matches were done on the patient on thioprine. Six patients were treated for AHR with plasmapheresis, antithymocyte globulin (patient 9). and/or mycophenolate mofetil and tacrolimus, as described (10). Cases from three other groups were compared. These cases were included if they met the diagnostic criteria for either: (1) acute cellular Immunofluorescence Microscopy rejection (14 patients) according to the Cooperative Clinical Trials in Biopsy sections were stained with a three-step immunofluores- Transplantation criteria (six type I, seven type II, and one type III) cence technique developed in our laboratory. Four-micrometer frozen (12); (2) acute CsA toxicity (six patients, including two cases with sections were incubated in 100 mg/ml avidin D (Vector Laboratories, thrombotic microangiopathy) (11); or (3) had no diagnostic abnor- Burlingame, CA) to block endogenous biotin. Sections were washed mality (four patients, biopsies taken at the time of transplantation). and excess avidin was bound by adding 10 mg/ml d-biotin (Sigma Cases were screened sequentially from the same time period as the Chemical Co., St. Louis, MO). Monoclonal antibody to C4d (clone AHR cases, starting with the most recent samples; cases with suffi- 10-11; Biogenesis, Sandown, NH) was applied for 30 min. Sections cient frozen tissue available for study were used. Biopsies were were washed and incubated sequentially first with biotinylated horse analyzed by light, immunofluorescence, and electron microscopy, as anti-mouse IgG (1:100) (Vector Laboratories) and after washing then described (13). with FITC-streptavidin (1:50) (Biomeda, Foster City, CA) each for 30 min. Endothelial cells were detected with biotinylated Ulex europaeus agglutinin-I (Ulex lectin, Vector Laboratories), which binds to a-L- Detection of Anti-Donor Antibodies fucose (in blood group substance O) (14). Donor T and B cells were isolated with immunomagnetic beads at For simultaneous detection of vascular basement membranes and transplantation and stored at 270°C (10). Serial serum samples were C4d, monoclonal anti-type IV collagen (Silenus, Hawthorne, Austra- collected prospectively. Cytotoxic cross-matches were performed us- lia) and tetramethylrhodamine isothiocyanate-rabbit anti-mouse IgG ing patient sera taken within1dofthebiopsy. Both anti-human (Dako, Carpinteria, CA) were added to the above C4d protocol. For globulin-enhanced T cell and standard complement-dependent cyto- simultaneous C4d and endothelial staining, C4d was detected with toxic B cell assays were used (10). Panel-reactive antibodies were tetramethylrhodamine isothiocyanate-rabbit anti-mouse IgG and Ulex determined with a local frozen cell panel. OKT3 was removed from lectin was detected with FITC-streptavidin. Table 1. Acute humoral rejection in patents with renal allograftsa Circulating Antibodies Donor to Donor Biopsy PTC PTC PTC PTC Graft Creatinine Patient b b (mg/dl) at Source No. PMN IgM C3 C4d Survival 1yr T Cells B Cells 1 CD 1:4 .1:8 1 (5) 21222to31 Failed (8) 2CD.1:8 .1:8 1 (8) 3112 41 Survived 1.6 3 CD, 3 ND ND 2 (17) 21221to21 Failed (180) .1:8 .1:8 3 (33) 2122 21 4c CD, 2 1:32 Negative 1 (6) 2121 31 Failed (23) 1:16 ND 2 (12) 2121/2 31 5 Daughter 1:32 .1:8 1 (7) 2122 41 Survived 1:16 ND 2 (34) 3122 41 1.8 6CD.1:8 1:4 2 (26) 2122 41 Survived 1.2 7 CD 1:128 1:4 1 (14) 2121 41 Survived .1:512 1:64 2 (21) 3122 41 1.1 8 CD, 3 Negative .1:8 1 (7) 1 to 212 2 41 Survived 1.3 9d CD, 3 Positivee Positivee 1 (3) 3111 41 Failed (10) 10 Mother Negative 1:256 1 (6) 3111 41 Survivedf 1:2 1:128 2 (17) 3121 31 Weak 1:16 3 (24) 2 to 312 1 2to31 3.1 a PTC, peritubular capillaries; PMN, polymorphonuclear leukocytes; ND, not determined; CD, cadaveric kidney. The number after CD indicates the number of renal allografts the patient has received. b The number in parentheses indicates days posttransplantation. c Patient was presensitized at time of transplantation. d This patient’s original disease was hereditary nephritis. His clinical course was complicated by the presence of anti-glomerular basement membrane antibodies in the kidney. e Determined by flow cytometry. f Graft has survived the first 6 wk. Patients 5 through 10 were treated with a rescue protocol combining tacrolimus with mycophenolate and/or plasma exchange (10). 2210 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2208–2214, 1999 Statistical Analyses Sections were examined in an Olympus BX60 vertical illumination fluorescence microscope and scored for C4d staining by two observ- ers without knowledge of the clinical or pathologic diagnoses.