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Intragraft events preceding chronic renal allograft rejection in a modified tolerance protocol
Kidney International, Vol. 58 (2000), pp. 2546–2558
Intragraft events preceding chronic renal allograft rejection in a modified tolerance protocol AKIRA SHIMIZU, KAZUHIKO YAMADA, DAVID H. SACHS, and ROBERT B. COLVIN Department of Pathology, Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA, and Department of Pathology, Nippon Medical School, Tokyo, Japan
Intragraft events preceding chronic renal allograft rejection in a modified tolerance protocol. Background. Inbred miniature swine treated for 12 days with high-dose cyclosporine A develop tolerance to histocompatibility complex (MHC) class I-mismatched renal allografts. When this protocol was modified by adding thymectomy before transplant, all animals developed acute rejection. Thereafter, by day 100, one half developed chronic rejection (progression group) and the other half recovered (recovery group). This provides an excellent experimental model to identify the mechanisms of chronic rejection as well as the early changes that may predict chronic rejection. Methods. We assessed the cellular infiltration, immune activation, humoral immunity, and cell- and antibody-mediated graft injury in the progression and the recovery groups. In addition, we also examined circulating donor reactive cytotoxic T lymphocyte (CTL) and antidonor antibody in both groups. Results. From days 8 to 18 after transplantation, the two groups were indistinguishable. Both showed acute rejection with endarteritis (type II); had IgG and IgM deposition in glomeruli and small vessels; had an infiltrate with similar numbers of T cells, proliferating (PCNA⫹) and activated (interleukin-2 receptor⫹) cells; and had a similar degree of parenchymal cell apoptosis [in situ DNA nick-end labeling (TUNEL)⫹]. However, by days 30 to 60, the two groups could be distinguished by several intragraft features. The recovery group became tolerant and had diminished T-cell infiltration, activation and proliferation, and no detectable antibody deposition. The number of TUNEL⫹-injured parenchymal cells decreased. In contrast, the progression group showed persistent cell infiltration with activation and proliferation. Significantly prominent TUNEL⫹ apoptotic parenchymal cells in tubules, glomeruli, peritubular capillaries and arteries were seen from day 30 to day 100. Circulating donor reactive CTL and antidonor class I IgG were detected in the progression group at higher levels than in the recovery group from days 30 to 60. Conclusion. In tolerance-induction protocols, unstable tolerance induction is associated with the persistent immunologic activation that mediates immunologic destruction of graft paKey words: apoptosis, anti-MHC class I antibody, transplantation, cytotoxic T cell, kidney rejection prediction, parenchymal cells, major histocompatibility complex, xenograft model. Received for publication December 23, 1999 and in revised form May 2, 2000 Accepted for publication June 20, 2000
2000 by the International Society of Nephrology
renchymal cells and chronic rejection. Certain of the described immunopathologic findings (activation, proliferation, apoptosis, and antibody deposition) may be useful in distinguishing the type of rejection, that is, whether the allograft will progress to chronic rejection or recovery.
Inbred miniature swine are the only large animal in which one can reproducibly study the effects of selective matching within the major histocompatibility complex (MHC) on parameters of transplantation [1, 2]. They also share many immunologic and physiologic properties with humans and are useful for preclinical studies [2, 3]. Our group demonstrated that inbred miniature swine treated with 12 days of high-dose cyclosporine A (CsA) develop tolerance to MHC class II-matched, class I-mismatched renal allografts [2, 4]. The thymus is necessary for rapid and stable tolerance induction in this model, presumably caused by central selection mechanisms [5, 6]. If the protocol is modified by adding thymectomy 21 to 42 days prior to transplantation, only peripheral mechanisms of tolerance can operate. The thymectomy model may be relevant to the usual adult human with an atrophic thymus. These thymectomized pigs develop prolonged graft dysfunction with acute rejection on days 8 to 18 [5, 6]. In 50% of animals, chronic rejection with graft dysfunction develops by day 100. In the remaining 50%, dysfunction improves gradually with the development of transplant tolerance, presumably by more effective peripheral mechanisms. Chronic rejection is one of the leading and intractable causes of renal allograft loss. Despite its devastating impact on graft survival, the pathogenesis of chronic rejection is still unclear and is believed to be multifactorial, including immunologic and nonimmunologic factors [7–10]. Chronic rejection is often preceded by acute rejection [11–13], however, not all acute rejection episodes lead to chronic rejection. The morphological and immunologic features that promote or predict progression of chronic rejection have not been defined. The present model was chosen because either progres-
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sion to chronic rejection or recovery from acute rejection occurs spontaneously after the same initial treatment. This model thus provides a novel opportunity to assess the key pathogenetic features that predict or accompany progression to chronic rejection after acute rejection. We compared the intragraft events in acute rejection and thereafter in those that develop chronic rejection and those that recovered, focusing on (1) the phenotypic characteristics, (2) activation, (3) proliferation, (4) apoptosis in graft infiltrating cells, and (5) humoral and cellmediated graft cell injury. METHODS Animals, surgery, and immunosuppression Transplant donors and recipients were selected from our herd of partially inbred Massachusetts General Hospital (MGH) miniature swine at five to seven months of age. The immunogenetic characteristics of this herd and of the intra-MHC recombinant haplotypes available have been described previously [1, 2]. Recombinants swine lymphocyte antigen (SLA)gg (class Ic/c, class IId/d) animals were donors, and SLAdd (class Id/d, class IId/d) animals were recipients of orthotopic kidney grafts, in order to achieve a transplantation of SLA class IImatched, 2-haplotype class I-mismatched kidneys, as described previously [4–6]. In all animals, a complete (N ⫽ 6) or partial (N ⫽ 2) thymectomy was carried out 21 to 42 days before kidney transplantation, as described previously [5, 6]. CsA was provided by Novartis Pharmaceutical Corp. (Hanover, NJ, USA), and was administered daily as a single infusion at a dose of 10 to 13 mg/kg (adjusted to maintain a blood level of 400 to 800 ng/mL) for 12 consecutive days, starting on the day of kidney transplantation. The plasma creatinine (Cr) level was monitored to indicate graft function. Based on clinical course, we divided the animals into two groups: one progressed to chronic rejection by day 100 (progression group), and the other recovered from acute rejection (recovery group). Four thymectomized (three complete and one partial) animals were in the progression group, and another four (three complete and one partial) animals were in the recovery group. The morphological and molecular markers were quantitated and compared in serial biopsies from grafts in the two groups. Histologic examination In both the progression and the recovery groups, sequential wedge kidney biopsies were performed on postoperative days 8, 11, 18, 30, 60, and 100 and at spontaneous death. For light microscopic examination, tissue was fixed in 10% buffered formalin and was embedded in paraffin. Hematoxylin and eosin (HE) and periodic acidSchiff (PAS) stains were performed for histologic exami-
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nation. The biopsies samples were diagnosed using National Institutes of Health-Cooperative Clinical Trials in Transplantation (NIH-CCTT) classification of renal allograft rejection [14–16]. To clarify the phenotypes of infiltrating cells, frozen sections were stained by the standard avidin-biotin-horseradish-peroxidase complex (ABC) technique [17]. Primary antibodies included anti-pig monoclonal antibodies MSA4 (IgG2a, anti-swine CD2), BB23-8E6 (IgG2b, antiswine CD3), 74-12-4 (IgG2b, anti-swine CD4), 76-2-11 (IgG2a, anti-swine CD8), BB6-11C9 (IgG1, anti-swine CD21; B cells), K231-3B2 [IgG1, anti-swine interleukin-2 receptor (IL2R)], and 74-22-15A (IgG1, macrophages) [18], and anti-human CD3 polyclonal antibodies (Dako, Glostrup, Denmark). The anti-human CD3 antibody was confirmed to react with swine pan T cells using swine thymus, lymph nodes, and spleen. For the detection of proliferating cell nuclear antigen (PCNA), 10% buffered formalin-fixed, paraffin-embedded tissue blocks were used, and sections were stained using ABC technique. In order to optimize detection of PCNA, microwave treatment (heat for 2 ⫻ 5 minutes in 0.01 mol/L sodium citrate, pH 6.0, in a 750 W microwave oven at full power and then immediately chilling to 4⬚C) and 1/1000 dilution of PC10 (IgG2a; Dako) was used [19]. Double immunostaining for PCNA and CD3 was performed in formalin-fixed paraffin sections using a two-color staining technique [17]. The sections were first stained with PCNA and incubated with alkaline phosphatase-labeled anti-mouse IgG (Vector, Burlingame, CA, USA) with a blue reaction product (Alkaline Phosphatase Substrate Kit III; Vector). Sections were then stained with polyclonal CD3, horseradish peroxidaselabeled anti-goat antibody (Dako), hydrogen peroxide (H2O2) containing 3,3⬘-diaminobendizine (DAB; Research Genetics, Hansville, AL, USA), which has a brown reaction product. Controls included omission or substitution of the primary antibodies with irrelevant antibodies. To detect antibody deposition in grafts, frozen tissue sections were stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-swine IgG or IgM (both from Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) and examined in an epifluorescence microscope (Zeiss, Oberkochen, Germany). In histologic sections, fragmented nuclear DNA associated with apoptosis and sometimes necrosis was labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method [20]. After deparaffinized and incubated with proteinase K 100 g/mL for 15 minutes, sections were rinsed in TdT buffer and incubated with TdT 1:25 and biotinylated-dUTP 1:20 in TdT buffer for 60 minutes at 37⬚C. The biotinylated nuclei were detected with avidin peroxidase and H2O2 containing DAB. Double immunostaining with
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TUNEL and CD3 for the identification of the origin of TUNEL⫹ cells was performed, by immunoalkaline phosphatase using TUNEL, followed by an antibody to CD3, horseradish peroxidase-labeled antigoat, and then incubated with H2O2 containing DAB. Controls consisted of omission of the dUTP or TdT. Quantitation of histologic findings Morphometric studies were performed to determine the number of CD2-, CD3-, CD4-, CD8-, or CD21-positive cells and macrophages per mm2, as well as the percentage of graft infiltrating cells that were PCNA⫹, IL2R⫹, or TUNEL⫹. In addition, the frequency of TUNEL⫹ graft parenchymal cells was measured in tubular epithelial cells, glomeruli, and peritubular and arterial endothelium, in order to detect ongoing antibody- and cell-mediated graft cell injury. More than 40 fields of renal cortex (at ⫻400, using an optical grid area of 0.0625 mm2) and more than 40 glomerular cross sections and all arterial cross sections in all fields of the renal cortex were counted in each kidney sample without prior knowledge of the clinical or histologic findings. Counts were expressed as the numbers of positive cells per mm2, the number of positive cells per glomerular cross section, the percentage of the infiltrating cells, or the percentage of the arteries affected. These results were expressed as the mean ⫾ SD or SEM, and statistical analysis was performed using the unpaired Student’s t-test. Cell-mediated lympholysis assay Cell-mediated lympholysis (CML) assays were performed, using peripheral blood leukocytes (PBLs), as described previously [4–6]. Briefly, lymphocyte cultures containing 4 ⫻ 106 responder and 4 ⫻ 106 irradiated (25G) stimulator PBL in 2 mL of medium were incubated for six days at 37⬚C in 7.5% CO2 and 100% humidity. Bulk cultures were harvested, and effector cells were tested on 51Cr-labeled blasts. The tests were run at serially diluted ratios (100:1, 50:1, 25:1, 12.5:1). After 5.5 hours of effector cell incubation with the 5 ⫻ 103 specific targets, supernatants were harvested, and 51Cr release was determined on a gamma counter (Micromedics, Huntsville, AL, USA). Maximum lysis was obtained with a 1% solution of the nonionic detergent NP-40 (BLR, Rockville, MD, USA). Baseline levels were measured as the rate of spontaneous release of 51Cr from 5 ⫻ 103 targets. The data were expressed as percentage of specific lysis: % specific lysis ⫽ experimental release (cpm) ⫺ spontaneous release (cpm) ⫻ 100 maximum release (cpm) ⫺ spontaneous release (cpm)
The results of the progression and the recovery groups were expressed as the mean ⫾ SD, and statistical analysis was performed using the unpaired Student’s t-test.
Fig. 1. Graft function in the progression (䊉) and the recovery (䊊) groups. In the progression group, four animals developed long-term loss of graft function. In the recovery group, four animals showed a transient elevation in plasma creatinine (Cr) 18 to 40 days post-transplant, but gradually improved and had stable renal function remaining thereafter.
Flow cytometry The presence of antidonor class I (SLA class Ic/c) IgM and IgG in the serum of experimental swine was detected by indirect flow cytometry using a Becton-Dickinson FACScan (Sunnyvale, CA, USA) [6] and recombinant SLA PBL to determine the SLA-binding specificity of the antibody. For staining, 1 ⫻ 106 cells per tube of recombinant SLA PBL or donor-type PBL (SLAgg, class Ic/c, class IId/d) were resuspended in Hank’s balanced salt solution (Life Technologies, Grand Island, NY, USA) containing 0.1% bovine serum albumin (BSA) and 0.05% NaN3, and incubated for 30 minutes at 4⬚C with decomplemented test sera. FITC-labeled goat anti-swine IgM or IgG polyclonal antibodies were used as secondary reagents (Pharmagen). After a final wash, cells were analyzed by flow cytometry using propidium iodide gating to exclude dead cells. Both normal pig serum and pretransplant sera from each respective experimental animal were used to assure specific binding. The data were expressed as median fluorescence intensity, and the results in the progression and the recovery groups were expressed as the mean ⫾ SD. RESULTS Of the eight animals that underwent complete (N ⫽ 6) or partial (N ⫽ 2) thymectomy 21 to 42 days before transplant, all developed graft dysfunction in the early phase (Fig. 1). Thereafter, a markedly different clinical course was observed in these animals. Four thymectomized (three complete and one partial) animals had prolonged graft dysfunction between days 18 and 40, but
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Fig. 2. Recovery group. Recovery from acute rejection (type II) is shown with PAS (⫻125; A–D) and CD3⫹ stains (⫻250; E–H) on days 18 (A and E), 30 (B and F), 60 (C and G), and 100 (D and H). A diffuse mononuclear cell and CD3⫹ cell infiltrate is seen with tubulitis, acute allograft glomerulopathy, and endarteritis at day 18. Thereafter, the T cells diminish, and allografts recover from acute rejection leaving minimal interstitial fibrosis by day 100.
gradually improved spontaneously and subsequently had stable renal function for a long time (⬎day 100, recovery group). The other four (three complete and one partial thymectomized) animals developed progressive renal dysfunction (progression group); two died from uremia with massive proteinuria on day 42 and day 51. The morphological and immunologic markers were examined in serial biopsies taken from grafts in the progression group and compared with grafts in the recovery group. Recovery group: Resolution of type II acute rejection Initially, the recovery group had diffuse mononuclear cells, including CD3⫹ cells, which infiltrated the interstitium, tubules, glomeruli, and small arteries on days 8 through 18 with tubulitis, acute allograft glomerulopathy, and endarteritis (Fig. 2A, E). However, the mononuclear cell and CD3⫹ cell infiltrate gradually diminished with less prominent tubulitis by day 30 (Fig. 2 B, F). Thereafter, acute allograft glomerulopathy and arterial lesions resolved with segmental glomerular hypercellularity, minimal interstitial fibrosis, and mononuclear cell infiltrate (Fig. 2C, D, G, H). All allografts showed normal arteries by day 100.
During the acute rejection in the recovery group, between day 8 and day 18, many graft infiltrating cells expressed PCNA (Fig. 3A). Double staining revealed CD3⫹ cells that frequently expressed PCNA (Fig. 3B), indicating that many infiltrating T cells were proliferating in the grafts. IL2R⫹ infiltrating cells were found diffusely in the cortex on day 18 (Fig. 3C). IgG and IgM deposition was found in glomeruli, small arteries, and focal peritubular capillaries (Fig. 3D). These findings indicated that T-cell– and possibly antibody-mediated acute vascular rejection occurred in the acute phase, even in the recovery group. However, during the resolution process, by day 60, PCNA⫹, PCNA⫹ CD3⫹, and IL2R⫹ graft infiltrating cells diminished rapidly (Fig. 3 E–G), and only rare deposition of IgM and IgG was then detectable in the grafts (Fig. 3H). Progression group: Evolution of acute rejection to chronic rejection In the progression group, a similar extent of acute rejection and CD3⫹ cell infiltration was observed in the early phase (Fig. 4A, E). The diffuse interstitial mononuclear cell and CD3⫹ cell infiltration continued to day 30
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Fig. 3. Recovery group. Proliferating cell nuclear antigen⫹ (PCNA⫹) cells (A and E ), PCNA and CD3⫹ cells (B and F ), interleukin-2 receptor⫹ (IL2R⫹) cells (C and G ), and immunoglobulin G (IgG) deposition (D and H) in renal allografts on days 18 (A–D) and 60 (E–H). Many proliferating and activated infiltrating cells, numerous proliferating T cells, and intense glomerular IgG deposition are seen on day 18; however, these progressively diminish after acute rejection by day 60 [A and E: PCNA stain, ⫻400; B and F: double stain with PCNA (black) and CD3 (brown), ⫻800; C and G: IL2R stain, ⫻500; D and H: IgG stain, ⫻400].
with the development of acute allograft glomerulopathy and endarteritis (Fig. 4B, F). Subsequently, the mononuclear cell and CD3⫹ infiltrate gradually resolved; however, the glomerulopathy, vasculopathy, and interstitial fibrosis progressed by day 60 (Fig. 4C, G). By day 100, the allografts had morphologic findings characteristic of chronic rejection (Fig. 4D, H). The grafts from the two dead animals also morphologically showed the process of chronic rejection. Mononuclear cell lineages were assessed by immunohistochemistry (Fig. 5). In the progression and the recovery groups, acute rejection with a similar number of T cells and macrophages occurred at the early phase. Thereafter, the numbers of macrophage and T-cell subsets (CD2, CD3, CD4, and CD8⫹ cells) were all significantly greater in the progression group between day 30 to day 100. The proportions of each T-cell subset were
equivalent in all time points in the progression and the recovery groups. A few CD21⫹ B cells infiltrated allografts in the progression group, and the number was not different from the recovery group. Prominent PCNA⫹, PCNA⫹ CD3⫹, and IL2R⫹ graft infiltrating cells were seen in grafts with acute type II rejection in the progression group (Fig. 6A–C), and these persisted during the development of chronic rejection (Fig. 6 E–G). Markers of proliferation (PCNA⫹) and activation (IL2R⫹) in graft infiltrating cells in the early phase (day 8 to day 18) were not significantly different in the progression and the recovery groups (Fig. 7A, B). Thereafter, significantly more PCNA⫹ and IL2R⫹ cells remained in the progression group (day 30 to day 100). In contrast to the recovery group, IgG deposition in glomeruli, small arteries, and focal peritubular capillaries continued through the chronic phase (Fig. 6D, H).
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Fig. 4. Progression group. The development of chronic rejection is shown with PAS (⫻125; A–D) and CD3 (⫻250; E–H) stains on days 18 (A and E), 30 (B and F), 60 (C and G), and 100 (D and H). Acute rejection (type II) occurs with a diffuse mononuclear cell and CD3⫹ cell infiltrate, tubulitis, acute allograft glomerulopathy, and endarteritis at day 18. The cell infiltrate continues with the development of allograft glomerulopathy and arteriopathy at day 30 to day 60. Subsequently, typical histologic chronic rejection develops by day 100.
TUNEL⫹ graft infiltrating cells and graft parenchymal cells Numerous TUNEL⫹ infiltrating cells, many of which also expressed CD3, were observed (Fig. 8) in the progression and the recovery groups. The TUNEL⫹ graft infiltrating cells peaked at day 18 and gradually decreased thereafter. The frequency of TUNEL⫹ infiltrating cells in the progression group was slightly higher than in the recovery group, although the difference was not statistically significant between day 8 to day 60. In both the progression and the recovery groups, prominent TUNEL⫹ parenchymal cells were seen associated with CD3⫹ cells in the lesions of tubulitis, allograft glomerulopathy, peritubular capillary, and endarteritis during acute rejection. Thereafter, in the progression group, numerous TUNEL⫹ cells remained in these lesions during the development of chronic rejection (Fig. 9A–D). In contrast, TUNEL⫹ cells in these sites progressively diminished in the recovery group by day 100 (Fig. 9
E–H). Significant differences in the number of TUNEL⫹ cells in tubules, glomeruli, peritubular capillaries, and arteries were evident at day 60 between the progression and the recovery groups (Fig. 10), indicating that active graft cell injury continued through the chronic phase in the progression group. Cell-mediated cytotoxicity In the recovery group, antidonor cytotoxic T lymphocyte (CTL) reactivity gradually decreased by day 100 (Fig. 11A). However, in the progression group, the CTL reactivity maintained similar levels to the pretransplant levels by day 60 and was significantly higher than in the recovery group. The last samples (day 100) showed only low levels of CML in both groups. Immunoglobulin response The presence of the antidonor specific class I antibody in serum by flow cytometric analysis was correlated with
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Fig. 5. Graft infiltrating cells. The graft (A) CD3⫹, (D) CD8⫹, (E ) CD4, and (B) CD2⫹ cell, and macrophage infiltrate continues in the progression group (䊉) and gradually resolves in the recovery group (䊊). The number of each subset (excluding CD21⫹ B cells, F ) in the progression group is significantly higher than in the recovery group between day 30 to day 100, but the proportion of each subset is similar. (C ) Macrophages. (A–F ) Values are expressed as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01; *** P ⬍ 0.001.
antibody deposition in the grafts (Fig. 11B, C). In both groups, transient antidonor IgM and IgG in serum was detected. Antidonor class I IgG in serum progressively decreased in the recovery group by day 60, whereas it was detected in serum through day 60 in the progression group. Later samples (day 100) had no detectable antibody in either group. DISCUSSION In this study, we demonstrated that persistent immunologic activation and immunologic injury of graft parenchymal cells (tubules, glomeruli, peritubular capillaries, and small arteries) distinguish those grafts that progress to chronic rejection. Chronic rejection, sometimes referred to as “chronic allograft nephropathy,” remains one of the most important causes of graft loss after the first year. However, the pathogenesis of chronic rejection remains unclear [7–10]. Recent studies have indicated that either antibody or T cells can incite the chronic
arteriopathy during the development of chronic rejection [7, 21–24]. However, more typical chronic rejection (at least in mice) is associated with the presence of humoral reactivity to the donor [24, 25]. Consistent with this hypothesis, in the present model, IgG deposition in the grafts persisted in the progression group, and antidonorspecific class I antibody remained in the circulation for 60 days. Although anticlass I antibody can mediate acute humoral rejection [26, 27], our results suggest that it is also associated with the development of chronic rejection. Substantial evidence indicates that T cells also play a critical role in the development of chronic rejection, on their cytotoxity and production of cytokines [21, 22, 28, 29]. Many strategies that inhibit the T-cell–mediated response also reduce arterial intimal thickening in chronic rejection [30–32]. Macrophages are believed to play a critical role in chronic rejection by their secretion of various products, including cytokines, oxygen radicals, and growth factors [33, 34]. The infiltration of greater
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Fig. 6. Progression group. PCNA⫹ cells (A and E ), PCNA and CD3⫹ cells (B and F ), IL2R⫹ cells (C and G), and IgG deposition (D and H) on days 18 (A–D) and 60 (E–H). Many proliferating and activated infiltrating cells, numerous proliferating T cells, and intense glomerular IgG deposition are seen on day 18. Thereafter, these continue by day 60 with the development of chronic rejection [A and E: PCNA stain, ⫻400; B and F: double stain with PCNA (block) and CD3 (brown), ⫻800; C and G: IL2R stain, ⫻500; D and H: IgG stain, ⫻400].
Fig. 7. Percentage of PCNA⫹ (A), IL2R⫹ (B), and TUNEL⫹ (C ) graft infiltrating cells in the progression (䊉) and the recovery (䊊) groups. Mononuclear cell proliferation and activation persist in the progression group by day 100, and these cells are significantly higher in the progression group between day 30 and day 100. The frequency of TUNEL⫹ infiltrating cells in the progression group is slightly higher than in the recovery group, although the difference is statistically significant only at day 100. (A and B) Values are expressed as mean ⫾ SD. (C) Values are expressed as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
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Fig. 8. Apoptosis in graft infiltrating cells in the progression group on day 60. TUNEL stain (⫻500; A) and double stain with TUNEL method (black) and CD3 (brown, ⫻800; B) show the numerous TUNEL⫹ apoptotic infiltrating cells (arrow) and TUNEL⫹ CD3⫹ apoptotic infiltrating T cells (arrow).
numbers of T cells and macrophages into the grafts in the progression group in our study is compatible with this view. However, the T-cell phenotype was similar in the progression and the recovery groups, so that the phenotypic characteristics of the infiltrate had neither prognostic or diagnostic significance. The extent of TUNEL⫹ apoptotic graft infiltrating cells had little or no diagnostic value in predicting chronic rejection, since the difference in the frequency of TUNEL⫹ infiltrating cells was not statistically significant between the progression and the recovery groups on day 8 to day 60. Apoptosis of graft infiltrating cells may regulate the number of infiltrating cells, including donor reactive T cells, and cell-mediated antigraft activity may associate with activation-induced and alloantigen-induced cell death of T lymphocytes [35]. In the present study, infiltrating T cells undergo apoptosis. However, the rate of proliferation, influx or efflux, rather than apoptosis, probably determines the net accumulation of T cells in the graft. Indeed, increased levels of PCNA and IL2R⫹ mononuclear cells were present during the development of chronic rejection. Recent reports demonstrate that activation and proliferation of graft infiltrating cells could be useful in differentiating between rejection and other causes of graft dysfunction [36–38]. We found that persistent IL2R⫹ and PCNA⫹ cells precede the development of chronic rejection. Therefore, analysis of proliferation and activation of infiltrating leukocytes may be more useful in differentiating between progressing to failure and recovering grafts after acute rejection. T-cell–mediated cytotoxicity probably plays an important role in allograft rejection by the destruction (cell
lysis and apoptosis) of MHC incompatible cells [39–41]. Antibody- and complement-mediated cell injury may also play an important role by lysis of target cells by terminal complement components [42, 43]. The TUNEL method can detect DNA fragmentation in the process of apoptosis and cell necrosis [20, 44]. Therefore, the TUNEL method may be quite practical in the detection of injured cells in graft by antibody- and cell-mediated rejection. In this study, we have demonstrated that significant and persistent injury of graft parenchymal cells (tubules, glomeruli, peritubular capillaries, and small arteries) was associated with the development of chronic rejection. Persistent immunologic cytotoxicity is probably a central mechanism in the pathogenesis of chronic rejection in this model. In both groups, a similar degree of acute rejection occurred between day 8 and day 18. Thereafter, significant differences in antibody deposition, the frequency of PCNA⫹ or IL2R⫹ leukocytes and TUNEL⫹ graft parenchymal cells were evident as early as day 30 to day 60 between the progressing and the recovery groups, suggesting that the analysis of sequential graft biopsies during the acute episode (day 8 to 18) and three weeks or more after an acute rejection episode may predict chronic rejection (or recovery) better than a biopsy only during the acute phase. As noted previously in this article, acute rejection is strongly related to the development of chronic rejection. However, not all acute rejection leads to chronic rejection. Our study of the recovery group demonstrated that the injured allografts gradually recovered even from type II acute rejection. This was associated with a reduction of antidonor class I antibody, resolution of graft infiltrating cells with immune activation, and diminished immunemediated graft cell injury. It is notable that recovery from severe acute rejection occurred with the development of tolerance, without exogenous additional immunosuppression. Even in the recovery group, the early phase had antidonor class I IgG production, prominent T-cell infiltrate with immune activation, and persistent immune-mediated graft injury, indicating that tolerance induction is incomplete, associated with a break of thymic mechanisms for tolerance by thymectomy. However, thymectomized animals still have peripheral mechanisms for tolerance [5, 6]. Peripheral tolerance may promote clonal deletion or anergy of alloreactive T cells and could be mediated by a change in cytokine milieu or by suppressive mechanisms. Indeed, in the present study, our results indicate that loss of T cells by apoptosis (T-cell deletion) and limiting the proliferation, activation, and cytotoxicy of infiltrating T cells (T-cell anergy) were evident in the grafts. In our class I-mismatched renal allograft model, even in the nonthymectomized animals, it is likely that an alteration in cytokine production plays an important role in the induction of tolerance, since
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Fig. 9. Double stain with TUNEL method (black) and CD3 (brown) in tubulitis (A and E ), glomerulopathy (B and F ), peritubular capillaries (C and G ), and endarteritis (D and H) in the progression (A–D) and the recovery (E–H) groups on day 60 (⫻900). In the progression group, numerous TUNEL⫹ cells (arrow) are observed with many CD3⫹ cell infiltration. However, less prominent CD3⫹ cells and TUNEL⫹ cells are seen in the recovery group.
inhibition of T-cell help (IL-2) by CsA leads to longterm tolerance [2, 4], and altered cytokine production consistent with differential activation of Th1 and Th2 cells has been demonstrated in renal tissue from allografts [45, 46]. The graft infiltrate also contains immunoregulatory cells important in the adaptation of the host to the graft [47]. Also, a recent report from the other laboratory shows that chronically rejected rat kidney allografts can paradoxically induce donor-specific tolerance [48]. In the recovery group, the mechanisms of selflimited cellular as well as humoral immunity and the development of tolerance during acute rejection are still unclear and are the subject of continuing investigations in our laboratory. However, it appears that cell- and antibody-mediated acute rejection may resolve during the development of transplant tolerance (without antirejection therapy), and injured graft associated with celland antibody-mediated rejection may recover to longterm functioning graft.
Recent studies in rodent and large animal models demonstrate that various strategies for tolerance induction prevent development of chronic rejection [49, 50]. In contrast, the progression group in our study suggests that if tolerance induction to allografts was delayed (or did not occur), persistent immunologic graft injury leads to chronic rejection. We therefore conclude that in treatment using the tolerant induction protocol, rapid and stable tolerance induction is important for long-term stable graft acceptance, before critical steps in the process of chronic rejection become established. Our results show that the immune activation resolved by day 60 in the recovery group, although active immune-mediated graft injury continued to at least day 60 in the progression group. Thus, day 60 after transplantation is a turning point for either recovery from rejection and long-term graft acceptance or progression to chronic rejection in this tolerance-induction protocol.
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Fig. 10. TUNEL⫹ cells in tubules (A), glomeruli (B), peritubular capillaries (C ), and arteries (D) in the progression (䊉) and the recovery (䊊) groups. TUNEL⫹ graft parenchymal cell injury continues in the progression group, and the number of TUNEL⫹ cells is significantly higher than in the recovery group on day 60. In the recovery group, TUNEL⫹ cells progressively reduce by day 100. Values are expressed as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
Fig. 11. Circulating anti-donor reactive cytotoxic T lymphocyte (CTL; A) and anti-donor class I IgM (B) and IgG (C ) in the progression (䊉) and the recovery (䊊) groups. Cell-mediated ML assay (A) shows that the anti-donor CTL reactivity is higher in the progression group by day 60 (effector:target ratio is 100:1). Flow cytometric analysis for detection of the anti-donor class I antibody (B and C) shows that transient anti-donor class I IgM and IgG in serum are seen in the recovery group. However, anti-donor class I IgG production continued through day 60 in the progression group. Values are expressed as mean ⫾ SD. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
Shimizu et al: Intragraft events preceding rejection
ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health: RO1-AI 31046, PO1-H218646, and PO1-HL 18646. Support was also received from the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C2642,4578). Portions of this study were previously published in abstract form (Shimizu et al, J Am Soc Nephrol 8:667A, 1997). The expert technical assistance of Ms. Patricia Della Pelle and Mr. Joseph Ambroz is gratefully acknowledged. Reprint requests to Robert B. Colvin, M.D., Department of Pathology, Massachusetts General Hospital, Warren 225, Boston, Massachusetts 02114, USA. E-mail: [email protected]
APPENDIX
13.
14. 15.
16. 17.
Abbreviations used in this article are: ABC, avidin-biotin-peroxidase complex; CD, cluster of differentiation; CML, cell-mediated lympholysis; Cr, creatinine; CsA, cyclosporine A; CTL, cytotoxic T lymphocyte; DAB, 3,3⬘-diaminobendizine; FITC, fluorescein isothiocyanate; HE, hematoxylin and eosin; H2O2, hydrogen peroxide; IL2R, interleukin 2 receptor; MHC, major histocompatibility complex, NIHCCTT, National Institutes of Health-Cooperative Clinical Trials in Transplantation; PAS, periodic acid-Schiff; PBL, peripheral blood lymphocytes; PCNA, proliferating cell nuclear antigen; SLA, swine leukocyte antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
19.
REFERENCES
21.
1. Sachs DH, Leight G, Cone J, Schwarz S, Stuart L, Rosenberg S: Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 22:559–567, 1976 2. Gianello P, Fishbein JM, Sachs DH: Tolerance to primary vascularized allografts in miniature swine. Immunol Rev 133:19–44, 1993 3. Pescovitz MD, Sachs DH, Lunney JK, Hsu SM: Localization of class II MHC antigen on porcine renal vascular endothelium. Transplantation 37:627–630, 1984 4. Rosengard BR, Ojikutu CA, Guzzetta PC, Smith CV, Sundt TM III, Nakajima K, Boorstein SM, Hill GS, Fishbein JM, Sachs DH: Induction of specific tolerance to class I-disparate renal allografts in miniature swine with cyclosporin. Transplantation 54:490– 497, 1992 5. Yamada K, Gianello PR, Ierino FL, Lorf T, Shimizu A, Meehan SM, Colvin RB, Sachs DH: Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts. J Exp Med 186:497–506, 1997 6. Yamada K, Ierino FL, Gianello PR, Shimizu A, Colvin RB, Sachs DH: Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I mismatched renal allografts. Transplantation 67:1112–1119, 1999 7. Tilney NL, Whitley WD, Diamond JR, Kupiec-Weglinski JW, Adams DH: Chronic rejection: An undefined conundrum. Transplantation 52:389–398, 1991 8. Ha¨yry P, Isoniemi H, Yilmaz S, Mennander A, Lemstro¨m K, Ra¨isa¨nen-Sokolwski A, Koskinen P, Ustinov J, Lautenschlager I, Taskinen E, Krogerus L, Aho P, Paavonen T: Chronic allograft rejection. Immunol Rev 134:33–81, 1993 9. Paul LC: Chronic renal transplant loss. Kidney Int 47:1491–1499, 1995 10. Kasiske BL: Clinical correlates to chronic renal allograft rejection. Kidney Int 52(Suppl 63):S71–S74, 1997 11. Almond PS, Matas A, Gillingham KJ, Dunn DL, Payne WD, Gores P, Gruessner R, Najarian JS: Risk factors for chronic rejection in renal allograft recipients. Transplantation 55:752–757, 1993 12. Yilmaz S, Ha¨yry P: The impact of acute episodes of rejection on
18.
20.
22.
23. 24.
25. 26.
27.
28. 29. 30.
31.
2557
the generation of chronic rejection in rat renal allografts. Transplantation 56:1153–1156, 1993 Van Saase JLCM, Van Der Woude FJ, Thorogood J, Hollander AAMJ, van Es LA, Eeening JJ, Van Bockel JH, Bruijn JA: The relation between acute vascular and interstitial renal allograft rejection and subsequent chronic rejection. Transplantation 59: 1280–1285, 1995 Colvin RB: The renal allograft biopsy. Kidney Int 50:1069–1082, 1996 Colvin RB, Cohen AH, Saiontz C, Bonsib S, Buick M, Burke B, Carter S, Cavallo T, Haas M, Lindblad A, Manivel CJ, Nast CC, Salomon D, Weaver C, Weiss M: Evaluation of pathological criteria for acute renal allograft rejection: Reproducibility, sensitivity and clinical correlation. J Am Soc Nephrol 8:1930–1941, 1997 Nickeleit V, Vamvakas EC, Pascual M, Poletti BJ, Colvin RB: The prognostic significance of specific arterial lesions in acute renal allograft rejection. J Am Soc Nephrol 9:1301–1308, 1998 Meehan SM, McCluskey RT, Pascual M, Preffer FI, Anderson P, Schlossman ST, Colvin RB: Cytotoxicity and apoptosis in human renal allografts: Identification, distribution, and quantitation of cells with a cytotoxic granule protein GMP-17 (TIA-1) and cells with fragmented nuclear DNA. Lab Invest 76:639–649, 1997 Saalmu¨ller A: Characterization of swine leukocyte differentiation antigens. Immunol Today 17:352–354, 1996 Lan HY, Mu W, Nikolic-Paterson DJ, Atkins RC: A novel, simple, reliable and sensitive method of multiple immunoenzymic staining: Use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J Histochem Cytochem 43:97–102, 1995 Gavrieli Y, Sherman Y, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501, 1992 Colvin RB, Chase CM, Winn HJ, Russell PS: Chronic allograft arteriopathy: Insights from experimental models, in Transplant Vascular Sclerosis, edited by Orosz C, Austin, Landes Biomedical Publishers, 1995, p. 7 Russell PS, Chase CM, Winn HJ, Colvin RB: Coronary atherosclerosis in transplanted mouse hearts. I. Time course and immunogenetic and immunopathological considerations. Am J Pathol 144:260–274, 1994 Russell PS, Chase CM, Winn HJ, Colvin RB: Coronary atherosclerosis in transplanted mouse hearts. II. Importance of humoral immunity. J Immunol 152:5135–5141, 1994 Hancock WH, Whitley WD, Tullius SG, Heemann UW, Wasowska B, Baldwin WM III, Tilney NL: Cytokines, adhesion molecules, and the pathogenesis of chronic rejection of rat renal allografts. Transplantation 56:643–650, 1993 Russell PS, Chase CM, Colvin RB: Alloantibody- and T cellmediated immunity in the pathogenesis of transplant arteriosclerosis. Transplantation 64:1531–1536, 1997 Trpkov K, Campbell P, Pazderka F, Cockfild S, Solz K, Halloran PF: Pathologic features of acute renal allograft rejection associated with donor-specific antibody. Transplantation 61:1586– 1592, 1996 Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-Rubin N, Cosimi AB, Colvin RB: Complement activation in acute humoral renal allograft rejection: Diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol 10:2208–2214, 1999 Nadeau KC, Azuma H, Tilney NL: Cytokines in the pathophysiology of acute and chronic allograft rejection. Transplant Rev 10:99– 107, 1996 Lemstro¨m K, Koskinen P, Ha¨yry P: Molecular mechanisms of chronic renal allograft rejection. Kidney Int 48(Suppl 52):S2–S10, 1995 Russell PS, Chase CM, Colvin RB: Coronary athrosclerosis in transplanted mouse hearts. IV. Effects of treatment with monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associated antigen-1. Transplantation 60:724–729, 1995 Koskinen PK, Lemstro¨m KB, Ha¨yry PJ: How cyclosporine modifies histological and molecular events in the vascular wall during chronic rejection of rat cardiac allografts. Am J Pathol 146:972–980, 1995
2558
Shimizu et al: Intragraft events preceding rejection
32. Russell ME, Hancock WW, Akalin E, Wallace AF, GlysingJensen T, Willett TA: Chronic cardiac rejection in the LEW to F344 rat model: Blockade of CD28-B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis. J Clin Invest 97:833–838, 1996 33. Russell ME, Wallace AF, Hancock WW, Sayegh MH, Adams DH, Sibinga NES, Wyner LR, Karnovsky MJ: Upregulation of cytokines associated with macrophage activation in the Lewisto-F344 rat transplantation model of chronic cardiac rejection. Transplantation 57:1367–1371, 1994 34. Croker BP, Clapp WL, Abu Shamat ARP, Kone BC, Peterson JC: Macrophages and chronic renal allograft nephropathy. Kidney Int 50(Suppl 57):S42–S49, 1996 35. Kabelitz D: Apoptosis, graft rejection, and transplantation tolerance. Transplantation 65:869–875, 1998 36. Sero´n D, Alexopoulos E, Raftery MJ, Hartley RB, Cameron JS: Diagnosis of rejection in renal allograft biopsies using the presence of activation and proliferating cells. Transplantation 47:811–816, 1989 37. Salom RN, Maguire JA, Esmore D, Hancock WW: Analysis of proliferating cell nuclear antigen expression aids histological diagnosis and is predictive of progression of human cardiac allograft rejection. Am J Pathol 145:876–882, 1994 38. Olsen S, Hansen HE: Proliferation rate of cells in the interstitial infiltrate in acute kidney allograft rejection. Transplant Proc 28: 502–503, 1996 39. Doherty PC: Cell-mediated cytotoxicity. Cell 75:607–612, 1993 40. Wever PC, Boonstra JG, Laterveer JC, Hack CE, Van Der Woude FJ, Daha MR, Ten Berge IJM: Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection. Transplantation 66:259–264, 1998 41. Ka¨gi D, Vignaux F, Ledermann BB, Bu¨rki K, Depraetere V, Nagata S, Hengartner H, Golstein P: Fas and perforin pathways
42. 43. 44.
45.
46.
47.
48.
49. 50.
as major mechanisms of T cell-mediated cytotoxicity. Science 265: 528–530, 1994 Quigg RJ: Mediation of glomerular injury: Glomerular injury induced by antibody and complement. Semin Nephrol 11:259–267, 1991 Hebert LA, Cosio FG, Birmingham DJ: The role of the complement system in renal injury. Semin Nephrol 12:408–427, 1992 Wijaman JH, Jonker RR, Keijzer R, Velde CJH, Cornelisse CJ, Dierendonck JH: A new method to detect apoptosis in paraffin sections: In situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7–12, 1993 Blancho G, Gianello P, Germana S, Baetscher M, Sachs DH, Leguern C: Molecular identification of porcine interleukin 10: Regulation of expression in a kidney allograft model. Proc Natl Acad Sci USA 92:2800–2804, 1995 Blancho G, Gianello P, Lorf T, Germana S, Giangrande I, Mourad G, Colvin RB, Sachs DH, Leguern C: Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine. Transplantation 63:26–33, 1997 Ierino FL, Yamada K, Hatch T, Rembert J, Sachs DH: Peripheral tolerance to class I mismatched renal allografts in miniature swine: Donor antigen-activated peripheral blood lymphocytes from tolerant swine inhibit anti-donor CTL reactivity. J Immunol 162:550– 559, 1999 Tullius SG, Nieminen M, Bechstein WO, Jonas S, Steinmu¨ller T, Pratschke J, Zeilinger K, Graser E, Volk HD, Neuhaus P: Chronically rejected rat kidney allografts induce donor-specific tolerance. Transplantation 64:158–161, 1997 Sayegh MH, Carpenter CB: Tolerance and chronic rejection. Kidney Int 51(Suppl 58):S11–S14, 1997 Madsen JC, Yamada K, Allan JS, Choo JK, erhorn AE, Pins MR, Vesga L, Slisz JK, Sachs DH: Transplantation tolerance prevents cardiac allograft vasculopathy in major histocompatibility complex class I-disparate miniature swine. Transplantation 65:304– 313, 1998
Intragraft events preceding chronic renal allograft rejection in a modified tolerance protocol AKIRA SHIMIZU, KAZUHIKO YAMADA, DAVID H. SACHS, and ROBERT B. COLVIN Department of Pathology, Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA, and Department of Pathology, Nippon Medical School, Tokyo, Japan
Intragraft events preceding chronic renal allograft rejection in a modified tolerance protocol. Background. Inbred miniature swine treated for 12 days with high-dose cyclosporine A develop tolerance to histocompatibility complex (MHC) class I-mismatched renal allografts. When this protocol was modified by adding thymectomy before transplant, all animals developed acute rejection. Thereafter, by day 100, one half developed chronic rejection (progression group) and the other half recovered (recovery group). This provides an excellent experimental model to identify the mechanisms of chronic rejection as well as the early changes that may predict chronic rejection. Methods. We assessed the cellular infiltration, immune activation, humoral immunity, and cell- and antibody-mediated graft injury in the progression and the recovery groups. In addition, we also examined circulating donor reactive cytotoxic T lymphocyte (CTL) and antidonor antibody in both groups. Results. From days 8 to 18 after transplantation, the two groups were indistinguishable. Both showed acute rejection with endarteritis (type II); had IgG and IgM deposition in glomeruli and small vessels; had an infiltrate with similar numbers of T cells, proliferating (PCNA⫹) and activated (interleukin-2 receptor⫹) cells; and had a similar degree of parenchymal cell apoptosis [in situ DNA nick-end labeling (TUNEL)⫹]. However, by days 30 to 60, the two groups could be distinguished by several intragraft features. The recovery group became tolerant and had diminished T-cell infiltration, activation and proliferation, and no detectable antibody deposition. The number of TUNEL⫹-injured parenchymal cells decreased. In contrast, the progression group showed persistent cell infiltration with activation and proliferation. Significantly prominent TUNEL⫹ apoptotic parenchymal cells in tubules, glomeruli, peritubular capillaries and arteries were seen from day 30 to day 100. Circulating donor reactive CTL and antidonor class I IgG were detected in the progression group at higher levels than in the recovery group from days 30 to 60. Conclusion. In tolerance-induction protocols, unstable tolerance induction is associated with the persistent immunologic activation that mediates immunologic destruction of graft paKey words: apoptosis, anti-MHC class I antibody, transplantation, cytotoxic T cell, kidney rejection prediction, parenchymal cells, major histocompatibility complex, xenograft model. Received for publication December 23, 1999 and in revised form May 2, 2000 Accepted for publication June 20, 2000
2000 by the International Society of Nephrology
renchymal cells and chronic rejection. Certain of the described immunopathologic findings (activation, proliferation, apoptosis, and antibody deposition) may be useful in distinguishing the type of rejection, that is, whether the allograft will progress to chronic rejection or recovery.
Inbred miniature swine are the only large animal in which one can reproducibly study the effects of selective matching within the major histocompatibility complex (MHC) on parameters of transplantation [1, 2]. They also share many immunologic and physiologic properties with humans and are useful for preclinical studies [2, 3]. Our group demonstrated that inbred miniature swine treated with 12 days of high-dose cyclosporine A (CsA) develop tolerance to MHC class II-matched, class I-mismatched renal allografts [2, 4]. The thymus is necessary for rapid and stable tolerance induction in this model, presumably caused by central selection mechanisms [5, 6]. If the protocol is modified by adding thymectomy 21 to 42 days prior to transplantation, only peripheral mechanisms of tolerance can operate. The thymectomy model may be relevant to the usual adult human with an atrophic thymus. These thymectomized pigs develop prolonged graft dysfunction with acute rejection on days 8 to 18 [5, 6]. In 50% of animals, chronic rejection with graft dysfunction develops by day 100. In the remaining 50%, dysfunction improves gradually with the development of transplant tolerance, presumably by more effective peripheral mechanisms. Chronic rejection is one of the leading and intractable causes of renal allograft loss. Despite its devastating impact on graft survival, the pathogenesis of chronic rejection is still unclear and is believed to be multifactorial, including immunologic and nonimmunologic factors [7–10]. Chronic rejection is often preceded by acute rejection [11–13], however, not all acute rejection episodes lead to chronic rejection. The morphological and immunologic features that promote or predict progression of chronic rejection have not been defined. The present model was chosen because either progres-
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sion to chronic rejection or recovery from acute rejection occurs spontaneously after the same initial treatment. This model thus provides a novel opportunity to assess the key pathogenetic features that predict or accompany progression to chronic rejection after acute rejection. We compared the intragraft events in acute rejection and thereafter in those that develop chronic rejection and those that recovered, focusing on (1) the phenotypic characteristics, (2) activation, (3) proliferation, (4) apoptosis in graft infiltrating cells, and (5) humoral and cellmediated graft cell injury. METHODS Animals, surgery, and immunosuppression Transplant donors and recipients were selected from our herd of partially inbred Massachusetts General Hospital (MGH) miniature swine at five to seven months of age. The immunogenetic characteristics of this herd and of the intra-MHC recombinant haplotypes available have been described previously [1, 2]. Recombinants swine lymphocyte antigen (SLA)gg (class Ic/c, class IId/d) animals were donors, and SLAdd (class Id/d, class IId/d) animals were recipients of orthotopic kidney grafts, in order to achieve a transplantation of SLA class IImatched, 2-haplotype class I-mismatched kidneys, as described previously [4–6]. In all animals, a complete (N ⫽ 6) or partial (N ⫽ 2) thymectomy was carried out 21 to 42 days before kidney transplantation, as described previously [5, 6]. CsA was provided by Novartis Pharmaceutical Corp. (Hanover, NJ, USA), and was administered daily as a single infusion at a dose of 10 to 13 mg/kg (adjusted to maintain a blood level of 400 to 800 ng/mL) for 12 consecutive days, starting on the day of kidney transplantation. The plasma creatinine (Cr) level was monitored to indicate graft function. Based on clinical course, we divided the animals into two groups: one progressed to chronic rejection by day 100 (progression group), and the other recovered from acute rejection (recovery group). Four thymectomized (three complete and one partial) animals were in the progression group, and another four (three complete and one partial) animals were in the recovery group. The morphological and molecular markers were quantitated and compared in serial biopsies from grafts in the two groups. Histologic examination In both the progression and the recovery groups, sequential wedge kidney biopsies were performed on postoperative days 8, 11, 18, 30, 60, and 100 and at spontaneous death. For light microscopic examination, tissue was fixed in 10% buffered formalin and was embedded in paraffin. Hematoxylin and eosin (HE) and periodic acidSchiff (PAS) stains were performed for histologic exami-
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nation. The biopsies samples were diagnosed using National Institutes of Health-Cooperative Clinical Trials in Transplantation (NIH-CCTT) classification of renal allograft rejection [14–16]. To clarify the phenotypes of infiltrating cells, frozen sections were stained by the standard avidin-biotin-horseradish-peroxidase complex (ABC) technique [17]. Primary antibodies included anti-pig monoclonal antibodies MSA4 (IgG2a, anti-swine CD2), BB23-8E6 (IgG2b, antiswine CD3), 74-12-4 (IgG2b, anti-swine CD4), 76-2-11 (IgG2a, anti-swine CD8), BB6-11C9 (IgG1, anti-swine CD21; B cells), K231-3B2 [IgG1, anti-swine interleukin-2 receptor (IL2R)], and 74-22-15A (IgG1, macrophages) [18], and anti-human CD3 polyclonal antibodies (Dako, Glostrup, Denmark). The anti-human CD3 antibody was confirmed to react with swine pan T cells using swine thymus, lymph nodes, and spleen. For the detection of proliferating cell nuclear antigen (PCNA), 10% buffered formalin-fixed, paraffin-embedded tissue blocks were used, and sections were stained using ABC technique. In order to optimize detection of PCNA, microwave treatment (heat for 2 ⫻ 5 minutes in 0.01 mol/L sodium citrate, pH 6.0, in a 750 W microwave oven at full power and then immediately chilling to 4⬚C) and 1/1000 dilution of PC10 (IgG2a; Dako) was used [19]. Double immunostaining for PCNA and CD3 was performed in formalin-fixed paraffin sections using a two-color staining technique [17]. The sections were first stained with PCNA and incubated with alkaline phosphatase-labeled anti-mouse IgG (Vector, Burlingame, CA, USA) with a blue reaction product (Alkaline Phosphatase Substrate Kit III; Vector). Sections were then stained with polyclonal CD3, horseradish peroxidaselabeled anti-goat antibody (Dako), hydrogen peroxide (H2O2) containing 3,3⬘-diaminobendizine (DAB; Research Genetics, Hansville, AL, USA), which has a brown reaction product. Controls included omission or substitution of the primary antibodies with irrelevant antibodies. To detect antibody deposition in grafts, frozen tissue sections were stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-swine IgG or IgM (both from Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) and examined in an epifluorescence microscope (Zeiss, Oberkochen, Germany). In histologic sections, fragmented nuclear DNA associated with apoptosis and sometimes necrosis was labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method [20]. After deparaffinized and incubated with proteinase K 100 g/mL for 15 minutes, sections were rinsed in TdT buffer and incubated with TdT 1:25 and biotinylated-dUTP 1:20 in TdT buffer for 60 minutes at 37⬚C. The biotinylated nuclei were detected with avidin peroxidase and H2O2 containing DAB. Double immunostaining with
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TUNEL and CD3 for the identification of the origin of TUNEL⫹ cells was performed, by immunoalkaline phosphatase using TUNEL, followed by an antibody to CD3, horseradish peroxidase-labeled antigoat, and then incubated with H2O2 containing DAB. Controls consisted of omission of the dUTP or TdT. Quantitation of histologic findings Morphometric studies were performed to determine the number of CD2-, CD3-, CD4-, CD8-, or CD21-positive cells and macrophages per mm2, as well as the percentage of graft infiltrating cells that were PCNA⫹, IL2R⫹, or TUNEL⫹. In addition, the frequency of TUNEL⫹ graft parenchymal cells was measured in tubular epithelial cells, glomeruli, and peritubular and arterial endothelium, in order to detect ongoing antibody- and cell-mediated graft cell injury. More than 40 fields of renal cortex (at ⫻400, using an optical grid area of 0.0625 mm2) and more than 40 glomerular cross sections and all arterial cross sections in all fields of the renal cortex were counted in each kidney sample without prior knowledge of the clinical or histologic findings. Counts were expressed as the numbers of positive cells per mm2, the number of positive cells per glomerular cross section, the percentage of the infiltrating cells, or the percentage of the arteries affected. These results were expressed as the mean ⫾ SD or SEM, and statistical analysis was performed using the unpaired Student’s t-test. Cell-mediated lympholysis assay Cell-mediated lympholysis (CML) assays were performed, using peripheral blood leukocytes (PBLs), as described previously [4–6]. Briefly, lymphocyte cultures containing 4 ⫻ 106 responder and 4 ⫻ 106 irradiated (25G) stimulator PBL in 2 mL of medium were incubated for six days at 37⬚C in 7.5% CO2 and 100% humidity. Bulk cultures were harvested, and effector cells were tested on 51Cr-labeled blasts. The tests were run at serially diluted ratios (100:1, 50:1, 25:1, 12.5:1). After 5.5 hours of effector cell incubation with the 5 ⫻ 103 specific targets, supernatants were harvested, and 51Cr release was determined on a gamma counter (Micromedics, Huntsville, AL, USA). Maximum lysis was obtained with a 1% solution of the nonionic detergent NP-40 (BLR, Rockville, MD, USA). Baseline levels were measured as the rate of spontaneous release of 51Cr from 5 ⫻ 103 targets. The data were expressed as percentage of specific lysis: % specific lysis ⫽ experimental release (cpm) ⫺ spontaneous release (cpm) ⫻ 100 maximum release (cpm) ⫺ spontaneous release (cpm)
The results of the progression and the recovery groups were expressed as the mean ⫾ SD, and statistical analysis was performed using the unpaired Student’s t-test.
Fig. 1. Graft function in the progression (䊉) and the recovery (䊊) groups. In the progression group, four animals developed long-term loss of graft function. In the recovery group, four animals showed a transient elevation in plasma creatinine (Cr) 18 to 40 days post-transplant, but gradually improved and had stable renal function remaining thereafter.
Flow cytometry The presence of antidonor class I (SLA class Ic/c) IgM and IgG in the serum of experimental swine was detected by indirect flow cytometry using a Becton-Dickinson FACScan (Sunnyvale, CA, USA) [6] and recombinant SLA PBL to determine the SLA-binding specificity of the antibody. For staining, 1 ⫻ 106 cells per tube of recombinant SLA PBL or donor-type PBL (SLAgg, class Ic/c, class IId/d) were resuspended in Hank’s balanced salt solution (Life Technologies, Grand Island, NY, USA) containing 0.1% bovine serum albumin (BSA) and 0.05% NaN3, and incubated for 30 minutes at 4⬚C with decomplemented test sera. FITC-labeled goat anti-swine IgM or IgG polyclonal antibodies were used as secondary reagents (Pharmagen). After a final wash, cells were analyzed by flow cytometry using propidium iodide gating to exclude dead cells. Both normal pig serum and pretransplant sera from each respective experimental animal were used to assure specific binding. The data were expressed as median fluorescence intensity, and the results in the progression and the recovery groups were expressed as the mean ⫾ SD. RESULTS Of the eight animals that underwent complete (N ⫽ 6) or partial (N ⫽ 2) thymectomy 21 to 42 days before transplant, all developed graft dysfunction in the early phase (Fig. 1). Thereafter, a markedly different clinical course was observed in these animals. Four thymectomized (three complete and one partial) animals had prolonged graft dysfunction between days 18 and 40, but
Shimizu et al: Intragraft events preceding rejection
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Fig. 2. Recovery group. Recovery from acute rejection (type II) is shown with PAS (⫻125; A–D) and CD3⫹ stains (⫻250; E–H) on days 18 (A and E), 30 (B and F), 60 (C and G), and 100 (D and H). A diffuse mononuclear cell and CD3⫹ cell infiltrate is seen with tubulitis, acute allograft glomerulopathy, and endarteritis at day 18. Thereafter, the T cells diminish, and allografts recover from acute rejection leaving minimal interstitial fibrosis by day 100.
gradually improved spontaneously and subsequently had stable renal function for a long time (⬎day 100, recovery group). The other four (three complete and one partial thymectomized) animals developed progressive renal dysfunction (progression group); two died from uremia with massive proteinuria on day 42 and day 51. The morphological and immunologic markers were examined in serial biopsies taken from grafts in the progression group and compared with grafts in the recovery group. Recovery group: Resolution of type II acute rejection Initially, the recovery group had diffuse mononuclear cells, including CD3⫹ cells, which infiltrated the interstitium, tubules, glomeruli, and small arteries on days 8 through 18 with tubulitis, acute allograft glomerulopathy, and endarteritis (Fig. 2A, E). However, the mononuclear cell and CD3⫹ cell infiltrate gradually diminished with less prominent tubulitis by day 30 (Fig. 2 B, F). Thereafter, acute allograft glomerulopathy and arterial lesions resolved with segmental glomerular hypercellularity, minimal interstitial fibrosis, and mononuclear cell infiltrate (Fig. 2C, D, G, H). All allografts showed normal arteries by day 100.
During the acute rejection in the recovery group, between day 8 and day 18, many graft infiltrating cells expressed PCNA (Fig. 3A). Double staining revealed CD3⫹ cells that frequently expressed PCNA (Fig. 3B), indicating that many infiltrating T cells were proliferating in the grafts. IL2R⫹ infiltrating cells were found diffusely in the cortex on day 18 (Fig. 3C). IgG and IgM deposition was found in glomeruli, small arteries, and focal peritubular capillaries (Fig. 3D). These findings indicated that T-cell– and possibly antibody-mediated acute vascular rejection occurred in the acute phase, even in the recovery group. However, during the resolution process, by day 60, PCNA⫹, PCNA⫹ CD3⫹, and IL2R⫹ graft infiltrating cells diminished rapidly (Fig. 3 E–G), and only rare deposition of IgM and IgG was then detectable in the grafts (Fig. 3H). Progression group: Evolution of acute rejection to chronic rejection In the progression group, a similar extent of acute rejection and CD3⫹ cell infiltration was observed in the early phase (Fig. 4A, E). The diffuse interstitial mononuclear cell and CD3⫹ cell infiltration continued to day 30
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Fig. 3. Recovery group. Proliferating cell nuclear antigen⫹ (PCNA⫹) cells (A and E ), PCNA and CD3⫹ cells (B and F ), interleukin-2 receptor⫹ (IL2R⫹) cells (C and G ), and immunoglobulin G (IgG) deposition (D and H) in renal allografts on days 18 (A–D) and 60 (E–H). Many proliferating and activated infiltrating cells, numerous proliferating T cells, and intense glomerular IgG deposition are seen on day 18; however, these progressively diminish after acute rejection by day 60 [A and E: PCNA stain, ⫻400; B and F: double stain with PCNA (black) and CD3 (brown), ⫻800; C and G: IL2R stain, ⫻500; D and H: IgG stain, ⫻400].
with the development of acute allograft glomerulopathy and endarteritis (Fig. 4B, F). Subsequently, the mononuclear cell and CD3⫹ infiltrate gradually resolved; however, the glomerulopathy, vasculopathy, and interstitial fibrosis progressed by day 60 (Fig. 4C, G). By day 100, the allografts had morphologic findings characteristic of chronic rejection (Fig. 4D, H). The grafts from the two dead animals also morphologically showed the process of chronic rejection. Mononuclear cell lineages were assessed by immunohistochemistry (Fig. 5). In the progression and the recovery groups, acute rejection with a similar number of T cells and macrophages occurred at the early phase. Thereafter, the numbers of macrophage and T-cell subsets (CD2, CD3, CD4, and CD8⫹ cells) were all significantly greater in the progression group between day 30 to day 100. The proportions of each T-cell subset were
equivalent in all time points in the progression and the recovery groups. A few CD21⫹ B cells infiltrated allografts in the progression group, and the number was not different from the recovery group. Prominent PCNA⫹, PCNA⫹ CD3⫹, and IL2R⫹ graft infiltrating cells were seen in grafts with acute type II rejection in the progression group (Fig. 6A–C), and these persisted during the development of chronic rejection (Fig. 6 E–G). Markers of proliferation (PCNA⫹) and activation (IL2R⫹) in graft infiltrating cells in the early phase (day 8 to day 18) were not significantly different in the progression and the recovery groups (Fig. 7A, B). Thereafter, significantly more PCNA⫹ and IL2R⫹ cells remained in the progression group (day 30 to day 100). In contrast to the recovery group, IgG deposition in glomeruli, small arteries, and focal peritubular capillaries continued through the chronic phase (Fig. 6D, H).
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Fig. 4. Progression group. The development of chronic rejection is shown with PAS (⫻125; A–D) and CD3 (⫻250; E–H) stains on days 18 (A and E), 30 (B and F), 60 (C and G), and 100 (D and H). Acute rejection (type II) occurs with a diffuse mononuclear cell and CD3⫹ cell infiltrate, tubulitis, acute allograft glomerulopathy, and endarteritis at day 18. The cell infiltrate continues with the development of allograft glomerulopathy and arteriopathy at day 30 to day 60. Subsequently, typical histologic chronic rejection develops by day 100.
TUNEL⫹ graft infiltrating cells and graft parenchymal cells Numerous TUNEL⫹ infiltrating cells, many of which also expressed CD3, were observed (Fig. 8) in the progression and the recovery groups. The TUNEL⫹ graft infiltrating cells peaked at day 18 and gradually decreased thereafter. The frequency of TUNEL⫹ infiltrating cells in the progression group was slightly higher than in the recovery group, although the difference was not statistically significant between day 8 to day 60. In both the progression and the recovery groups, prominent TUNEL⫹ parenchymal cells were seen associated with CD3⫹ cells in the lesions of tubulitis, allograft glomerulopathy, peritubular capillary, and endarteritis during acute rejection. Thereafter, in the progression group, numerous TUNEL⫹ cells remained in these lesions during the development of chronic rejection (Fig. 9A–D). In contrast, TUNEL⫹ cells in these sites progressively diminished in the recovery group by day 100 (Fig. 9
E–H). Significant differences in the number of TUNEL⫹ cells in tubules, glomeruli, peritubular capillaries, and arteries were evident at day 60 between the progression and the recovery groups (Fig. 10), indicating that active graft cell injury continued through the chronic phase in the progression group. Cell-mediated cytotoxicity In the recovery group, antidonor cytotoxic T lymphocyte (CTL) reactivity gradually decreased by day 100 (Fig. 11A). However, in the progression group, the CTL reactivity maintained similar levels to the pretransplant levels by day 60 and was significantly higher than in the recovery group. The last samples (day 100) showed only low levels of CML in both groups. Immunoglobulin response The presence of the antidonor specific class I antibody in serum by flow cytometric analysis was correlated with
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Fig. 5. Graft infiltrating cells. The graft (A) CD3⫹, (D) CD8⫹, (E ) CD4, and (B) CD2⫹ cell, and macrophage infiltrate continues in the progression group (䊉) and gradually resolves in the recovery group (䊊). The number of each subset (excluding CD21⫹ B cells, F ) in the progression group is significantly higher than in the recovery group between day 30 to day 100, but the proportion of each subset is similar. (C ) Macrophages. (A–F ) Values are expressed as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01; *** P ⬍ 0.001.
antibody deposition in the grafts (Fig. 11B, C). In both groups, transient antidonor IgM and IgG in serum was detected. Antidonor class I IgG in serum progressively decreased in the recovery group by day 60, whereas it was detected in serum through day 60 in the progression group. Later samples (day 100) had no detectable antibody in either group. DISCUSSION In this study, we demonstrated that persistent immunologic activation and immunologic injury of graft parenchymal cells (tubules, glomeruli, peritubular capillaries, and small arteries) distinguish those grafts that progress to chronic rejection. Chronic rejection, sometimes referred to as “chronic allograft nephropathy,” remains one of the most important causes of graft loss after the first year. However, the pathogenesis of chronic rejection remains unclear [7–10]. Recent studies have indicated that either antibody or T cells can incite the chronic
arteriopathy during the development of chronic rejection [7, 21–24]. However, more typical chronic rejection (at least in mice) is associated with the presence of humoral reactivity to the donor [24, 25]. Consistent with this hypothesis, in the present model, IgG deposition in the grafts persisted in the progression group, and antidonorspecific class I antibody remained in the circulation for 60 days. Although anticlass I antibody can mediate acute humoral rejection [26, 27], our results suggest that it is also associated with the development of chronic rejection. Substantial evidence indicates that T cells also play a critical role in the development of chronic rejection, on their cytotoxity and production of cytokines [21, 22, 28, 29]. Many strategies that inhibit the T-cell–mediated response also reduce arterial intimal thickening in chronic rejection [30–32]. Macrophages are believed to play a critical role in chronic rejection by their secretion of various products, including cytokines, oxygen radicals, and growth factors [33, 34]. The infiltration of greater
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Fig. 6. Progression group. PCNA⫹ cells (A and E ), PCNA and CD3⫹ cells (B and F ), IL2R⫹ cells (C and G), and IgG deposition (D and H) on days 18 (A–D) and 60 (E–H). Many proliferating and activated infiltrating cells, numerous proliferating T cells, and intense glomerular IgG deposition are seen on day 18. Thereafter, these continue by day 60 with the development of chronic rejection [A and E: PCNA stain, ⫻400; B and F: double stain with PCNA (block) and CD3 (brown), ⫻800; C and G: IL2R stain, ⫻500; D and H: IgG stain, ⫻400].
Fig. 7. Percentage of PCNA⫹ (A), IL2R⫹ (B), and TUNEL⫹ (C ) graft infiltrating cells in the progression (䊉) and the recovery (䊊) groups. Mononuclear cell proliferation and activation persist in the progression group by day 100, and these cells are significantly higher in the progression group between day 30 and day 100. The frequency of TUNEL⫹ infiltrating cells in the progression group is slightly higher than in the recovery group, although the difference is statistically significant only at day 100. (A and B) Values are expressed as mean ⫾ SD. (C) Values are expressed as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
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Fig. 8. Apoptosis in graft infiltrating cells in the progression group on day 60. TUNEL stain (⫻500; A) and double stain with TUNEL method (black) and CD3 (brown, ⫻800; B) show the numerous TUNEL⫹ apoptotic infiltrating cells (arrow) and TUNEL⫹ CD3⫹ apoptotic infiltrating T cells (arrow).
numbers of T cells and macrophages into the grafts in the progression group in our study is compatible with this view. However, the T-cell phenotype was similar in the progression and the recovery groups, so that the phenotypic characteristics of the infiltrate had neither prognostic or diagnostic significance. The extent of TUNEL⫹ apoptotic graft infiltrating cells had little or no diagnostic value in predicting chronic rejection, since the difference in the frequency of TUNEL⫹ infiltrating cells was not statistically significant between the progression and the recovery groups on day 8 to day 60. Apoptosis of graft infiltrating cells may regulate the number of infiltrating cells, including donor reactive T cells, and cell-mediated antigraft activity may associate with activation-induced and alloantigen-induced cell death of T lymphocytes [35]. In the present study, infiltrating T cells undergo apoptosis. However, the rate of proliferation, influx or efflux, rather than apoptosis, probably determines the net accumulation of T cells in the graft. Indeed, increased levels of PCNA and IL2R⫹ mononuclear cells were present during the development of chronic rejection. Recent reports demonstrate that activation and proliferation of graft infiltrating cells could be useful in differentiating between rejection and other causes of graft dysfunction [36–38]. We found that persistent IL2R⫹ and PCNA⫹ cells precede the development of chronic rejection. Therefore, analysis of proliferation and activation of infiltrating leukocytes may be more useful in differentiating between progressing to failure and recovering grafts after acute rejection. T-cell–mediated cytotoxicity probably plays an important role in allograft rejection by the destruction (cell
lysis and apoptosis) of MHC incompatible cells [39–41]. Antibody- and complement-mediated cell injury may also play an important role by lysis of target cells by terminal complement components [42, 43]. The TUNEL method can detect DNA fragmentation in the process of apoptosis and cell necrosis [20, 44]. Therefore, the TUNEL method may be quite practical in the detection of injured cells in graft by antibody- and cell-mediated rejection. In this study, we have demonstrated that significant and persistent injury of graft parenchymal cells (tubules, glomeruli, peritubular capillaries, and small arteries) was associated with the development of chronic rejection. Persistent immunologic cytotoxicity is probably a central mechanism in the pathogenesis of chronic rejection in this model. In both groups, a similar degree of acute rejection occurred between day 8 and day 18. Thereafter, significant differences in antibody deposition, the frequency of PCNA⫹ or IL2R⫹ leukocytes and TUNEL⫹ graft parenchymal cells were evident as early as day 30 to day 60 between the progressing and the recovery groups, suggesting that the analysis of sequential graft biopsies during the acute episode (day 8 to 18) and three weeks or more after an acute rejection episode may predict chronic rejection (or recovery) better than a biopsy only during the acute phase. As noted previously in this article, acute rejection is strongly related to the development of chronic rejection. However, not all acute rejection leads to chronic rejection. Our study of the recovery group demonstrated that the injured allografts gradually recovered even from type II acute rejection. This was associated with a reduction of antidonor class I antibody, resolution of graft infiltrating cells with immune activation, and diminished immunemediated graft cell injury. It is notable that recovery from severe acute rejection occurred with the development of tolerance, without exogenous additional immunosuppression. Even in the recovery group, the early phase had antidonor class I IgG production, prominent T-cell infiltrate with immune activation, and persistent immune-mediated graft injury, indicating that tolerance induction is incomplete, associated with a break of thymic mechanisms for tolerance by thymectomy. However, thymectomized animals still have peripheral mechanisms for tolerance [5, 6]. Peripheral tolerance may promote clonal deletion or anergy of alloreactive T cells and could be mediated by a change in cytokine milieu or by suppressive mechanisms. Indeed, in the present study, our results indicate that loss of T cells by apoptosis (T-cell deletion) and limiting the proliferation, activation, and cytotoxicy of infiltrating T cells (T-cell anergy) were evident in the grafts. In our class I-mismatched renal allograft model, even in the nonthymectomized animals, it is likely that an alteration in cytokine production plays an important role in the induction of tolerance, since
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Fig. 9. Double stain with TUNEL method (black) and CD3 (brown) in tubulitis (A and E ), glomerulopathy (B and F ), peritubular capillaries (C and G ), and endarteritis (D and H) in the progression (A–D) and the recovery (E–H) groups on day 60 (⫻900). In the progression group, numerous TUNEL⫹ cells (arrow) are observed with many CD3⫹ cell infiltration. However, less prominent CD3⫹ cells and TUNEL⫹ cells are seen in the recovery group.
inhibition of T-cell help (IL-2) by CsA leads to longterm tolerance [2, 4], and altered cytokine production consistent with differential activation of Th1 and Th2 cells has been demonstrated in renal tissue from allografts [45, 46]. The graft infiltrate also contains immunoregulatory cells important in the adaptation of the host to the graft [47]. Also, a recent report from the other laboratory shows that chronically rejected rat kidney allografts can paradoxically induce donor-specific tolerance [48]. In the recovery group, the mechanisms of selflimited cellular as well as humoral immunity and the development of tolerance during acute rejection are still unclear and are the subject of continuing investigations in our laboratory. However, it appears that cell- and antibody-mediated acute rejection may resolve during the development of transplant tolerance (without antirejection therapy), and injured graft associated with celland antibody-mediated rejection may recover to longterm functioning graft.
Recent studies in rodent and large animal models demonstrate that various strategies for tolerance induction prevent development of chronic rejection [49, 50]. In contrast, the progression group in our study suggests that if tolerance induction to allografts was delayed (or did not occur), persistent immunologic graft injury leads to chronic rejection. We therefore conclude that in treatment using the tolerant induction protocol, rapid and stable tolerance induction is important for long-term stable graft acceptance, before critical steps in the process of chronic rejection become established. Our results show that the immune activation resolved by day 60 in the recovery group, although active immune-mediated graft injury continued to at least day 60 in the progression group. Thus, day 60 after transplantation is a turning point for either recovery from rejection and long-term graft acceptance or progression to chronic rejection in this tolerance-induction protocol.
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Fig. 10. TUNEL⫹ cells in tubules (A), glomeruli (B), peritubular capillaries (C ), and arteries (D) in the progression (䊉) and the recovery (䊊) groups. TUNEL⫹ graft parenchymal cell injury continues in the progression group, and the number of TUNEL⫹ cells is significantly higher than in the recovery group on day 60. In the recovery group, TUNEL⫹ cells progressively reduce by day 100. Values are expressed as mean ⫾ SEM. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
Fig. 11. Circulating anti-donor reactive cytotoxic T lymphocyte (CTL; A) and anti-donor class I IgM (B) and IgG (C ) in the progression (䊉) and the recovery (䊊) groups. Cell-mediated ML assay (A) shows that the anti-donor CTL reactivity is higher in the progression group by day 60 (effector:target ratio is 100:1). Flow cytometric analysis for detection of the anti-donor class I antibody (B and C) shows that transient anti-donor class I IgM and IgG in serum are seen in the recovery group. However, anti-donor class I IgG production continued through day 60 in the progression group. Values are expressed as mean ⫾ SD. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
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ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health: RO1-AI 31046, PO1-H218646, and PO1-HL 18646. Support was also received from the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C2642,4578). Portions of this study were previously published in abstract form (Shimizu et al, J Am Soc Nephrol 8:667A, 1997). The expert technical assistance of Ms. Patricia Della Pelle and Mr. Joseph Ambroz is gratefully acknowledged. Reprint requests to Robert B. Colvin, M.D., Department of Pathology, Massachusetts General Hospital, Warren 225, Boston, Massachusetts 02114, USA. E-mail: [email protected]
APPENDIX
13.
14. 15.
16. 17.
Abbreviations used in this article are: ABC, avidin-biotin-peroxidase complex; CD, cluster of differentiation; CML, cell-mediated lympholysis; Cr, creatinine; CsA, cyclosporine A; CTL, cytotoxic T lymphocyte; DAB, 3,3⬘-diaminobendizine; FITC, fluorescein isothiocyanate; HE, hematoxylin and eosin; H2O2, hydrogen peroxide; IL2R, interleukin 2 receptor; MHC, major histocompatibility complex, NIHCCTT, National Institutes of Health-Cooperative Clinical Trials in Transplantation; PAS, periodic acid-Schiff; PBL, peripheral blood lymphocytes; PCNA, proliferating cell nuclear antigen; SLA, swine leukocyte antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
19.
REFERENCES
21.
1. Sachs DH, Leight G, Cone J, Schwarz S, Stuart L, Rosenberg S: Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 22:559–567, 1976 2. Gianello P, Fishbein JM, Sachs DH: Tolerance to primary vascularized allografts in miniature swine. Immunol Rev 133:19–44, 1993 3. Pescovitz MD, Sachs DH, Lunney JK, Hsu SM: Localization of class II MHC antigen on porcine renal vascular endothelium. Transplantation 37:627–630, 1984 4. Rosengard BR, Ojikutu CA, Guzzetta PC, Smith CV, Sundt TM III, Nakajima K, Boorstein SM, Hill GS, Fishbein JM, Sachs DH: Induction of specific tolerance to class I-disparate renal allografts in miniature swine with cyclosporin. Transplantation 54:490– 497, 1992 5. Yamada K, Gianello PR, Ierino FL, Lorf T, Shimizu A, Meehan SM, Colvin RB, Sachs DH: Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts. J Exp Med 186:497–506, 1997 6. Yamada K, Ierino FL, Gianello PR, Shimizu A, Colvin RB, Sachs DH: Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I mismatched renal allografts. Transplantation 67:1112–1119, 1999 7. Tilney NL, Whitley WD, Diamond JR, Kupiec-Weglinski JW, Adams DH: Chronic rejection: An undefined conundrum. Transplantation 52:389–398, 1991 8. Ha¨yry P, Isoniemi H, Yilmaz S, Mennander A, Lemstro¨m K, Ra¨isa¨nen-Sokolwski A, Koskinen P, Ustinov J, Lautenschlager I, Taskinen E, Krogerus L, Aho P, Paavonen T: Chronic allograft rejection. Immunol Rev 134:33–81, 1993 9. Paul LC: Chronic renal transplant loss. Kidney Int 47:1491–1499, 1995 10. Kasiske BL: Clinical correlates to chronic renal allograft rejection. Kidney Int 52(Suppl 63):S71–S74, 1997 11. Almond PS, Matas A, Gillingham KJ, Dunn DL, Payne WD, Gores P, Gruessner R, Najarian JS: Risk factors for chronic rejection in renal allograft recipients. Transplantation 55:752–757, 1993 12. Yilmaz S, Ha¨yry P: The impact of acute episodes of rejection on
18.
20.
22.
23. 24.
25. 26.
27.
28. 29. 30.
31.
2557
the generation of chronic rejection in rat renal allografts. Transplantation 56:1153–1156, 1993 Van Saase JLCM, Van Der Woude FJ, Thorogood J, Hollander AAMJ, van Es LA, Eeening JJ, Van Bockel JH, Bruijn JA: The relation between acute vascular and interstitial renal allograft rejection and subsequent chronic rejection. Transplantation 59: 1280–1285, 1995 Colvin RB: The renal allograft biopsy. Kidney Int 50:1069–1082, 1996 Colvin RB, Cohen AH, Saiontz C, Bonsib S, Buick M, Burke B, Carter S, Cavallo T, Haas M, Lindblad A, Manivel CJ, Nast CC, Salomon D, Weaver C, Weiss M: Evaluation of pathological criteria for acute renal allograft rejection: Reproducibility, sensitivity and clinical correlation. J Am Soc Nephrol 8:1930–1941, 1997 Nickeleit V, Vamvakas EC, Pascual M, Poletti BJ, Colvin RB: The prognostic significance of specific arterial lesions in acute renal allograft rejection. J Am Soc Nephrol 9:1301–1308, 1998 Meehan SM, McCluskey RT, Pascual M, Preffer FI, Anderson P, Schlossman ST, Colvin RB: Cytotoxicity and apoptosis in human renal allografts: Identification, distribution, and quantitation of cells with a cytotoxic granule protein GMP-17 (TIA-1) and cells with fragmented nuclear DNA. Lab Invest 76:639–649, 1997 Saalmu¨ller A: Characterization of swine leukocyte differentiation antigens. Immunol Today 17:352–354, 1996 Lan HY, Mu W, Nikolic-Paterson DJ, Atkins RC: A novel, simple, reliable and sensitive method of multiple immunoenzymic staining: Use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J Histochem Cytochem 43:97–102, 1995 Gavrieli Y, Sherman Y, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501, 1992 Colvin RB, Chase CM, Winn HJ, Russell PS: Chronic allograft arteriopathy: Insights from experimental models, in Transplant Vascular Sclerosis, edited by Orosz C, Austin, Landes Biomedical Publishers, 1995, p. 7 Russell PS, Chase CM, Winn HJ, Colvin RB: Coronary atherosclerosis in transplanted mouse hearts. I. Time course and immunogenetic and immunopathological considerations. Am J Pathol 144:260–274, 1994 Russell PS, Chase CM, Winn HJ, Colvin RB: Coronary atherosclerosis in transplanted mouse hearts. II. Importance of humoral immunity. J Immunol 152:5135–5141, 1994 Hancock WH, Whitley WD, Tullius SG, Heemann UW, Wasowska B, Baldwin WM III, Tilney NL: Cytokines, adhesion molecules, and the pathogenesis of chronic rejection of rat renal allografts. Transplantation 56:643–650, 1993 Russell PS, Chase CM, Colvin RB: Alloantibody- and T cellmediated immunity in the pathogenesis of transplant arteriosclerosis. Transplantation 64:1531–1536, 1997 Trpkov K, Campbell P, Pazderka F, Cockfild S, Solz K, Halloran PF: Pathologic features of acute renal allograft rejection associated with donor-specific antibody. Transplantation 61:1586– 1592, 1996 Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-Rubin N, Cosimi AB, Colvin RB: Complement activation in acute humoral renal allograft rejection: Diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol 10:2208–2214, 1999 Nadeau KC, Azuma H, Tilney NL: Cytokines in the pathophysiology of acute and chronic allograft rejection. Transplant Rev 10:99– 107, 1996 Lemstro¨m K, Koskinen P, Ha¨yry P: Molecular mechanisms of chronic renal allograft rejection. Kidney Int 48(Suppl 52):S2–S10, 1995 Russell PS, Chase CM, Colvin RB: Coronary athrosclerosis in transplanted mouse hearts. IV. Effects of treatment with monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associated antigen-1. Transplantation 60:724–729, 1995 Koskinen PK, Lemstro¨m KB, Ha¨yry PJ: How cyclosporine modifies histological and molecular events in the vascular wall during chronic rejection of rat cardiac allografts. Am J Pathol 146:972–980, 1995
2558
Shimizu et al: Intragraft events preceding rejection
32. Russell ME, Hancock WW, Akalin E, Wallace AF, GlysingJensen T, Willett TA: Chronic cardiac rejection in the LEW to F344 rat model: Blockade of CD28-B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis. J Clin Invest 97:833–838, 1996 33. Russell ME, Wallace AF, Hancock WW, Sayegh MH, Adams DH, Sibinga NES, Wyner LR, Karnovsky MJ: Upregulation of cytokines associated with macrophage activation in the Lewisto-F344 rat transplantation model of chronic cardiac rejection. Transplantation 57:1367–1371, 1994 34. Croker BP, Clapp WL, Abu Shamat ARP, Kone BC, Peterson JC: Macrophages and chronic renal allograft nephropathy. Kidney Int 50(Suppl 57):S42–S49, 1996 35. Kabelitz D: Apoptosis, graft rejection, and transplantation tolerance. Transplantation 65:869–875, 1998 36. Sero´n D, Alexopoulos E, Raftery MJ, Hartley RB, Cameron JS: Diagnosis of rejection in renal allograft biopsies using the presence of activation and proliferating cells. Transplantation 47:811–816, 1989 37. Salom RN, Maguire JA, Esmore D, Hancock WW: Analysis of proliferating cell nuclear antigen expression aids histological diagnosis and is predictive of progression of human cardiac allograft rejection. Am J Pathol 145:876–882, 1994 38. Olsen S, Hansen HE: Proliferation rate of cells in the interstitial infiltrate in acute kidney allograft rejection. Transplant Proc 28: 502–503, 1996 39. Doherty PC: Cell-mediated cytotoxicity. Cell 75:607–612, 1993 40. Wever PC, Boonstra JG, Laterveer JC, Hack CE, Van Der Woude FJ, Daha MR, Ten Berge IJM: Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection. Transplantation 66:259–264, 1998 41. Ka¨gi D, Vignaux F, Ledermann BB, Bu¨rki K, Depraetere V, Nagata S, Hengartner H, Golstein P: Fas and perforin pathways
42. 43. 44.
45.
46.
47.
48.
49. 50.
as major mechanisms of T cell-mediated cytotoxicity. Science 265: 528–530, 1994 Quigg RJ: Mediation of glomerular injury: Glomerular injury induced by antibody and complement. Semin Nephrol 11:259–267, 1991 Hebert LA, Cosio FG, Birmingham DJ: The role of the complement system in renal injury. Semin Nephrol 12:408–427, 1992 Wijaman JH, Jonker RR, Keijzer R, Velde CJH, Cornelisse CJ, Dierendonck JH: A new method to detect apoptosis in paraffin sections: In situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7–12, 1993 Blancho G, Gianello P, Germana S, Baetscher M, Sachs DH, Leguern C: Molecular identification of porcine interleukin 10: Regulation of expression in a kidney allograft model. Proc Natl Acad Sci USA 92:2800–2804, 1995 Blancho G, Gianello P, Lorf T, Germana S, Giangrande I, Mourad G, Colvin RB, Sachs DH, Leguern C: Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine. Transplantation 63:26–33, 1997 Ierino FL, Yamada K, Hatch T, Rembert J, Sachs DH: Peripheral tolerance to class I mismatched renal allografts in miniature swine: Donor antigen-activated peripheral blood lymphocytes from tolerant swine inhibit anti-donor CTL reactivity. J Immunol 162:550– 559, 1999 Tullius SG, Nieminen M, Bechstein WO, Jonas S, Steinmu¨ller T, Pratschke J, Zeilinger K, Graser E, Volk HD, Neuhaus P: Chronically rejected rat kidney allografts induce donor-specific tolerance. Transplantation 64:158–161, 1997 Sayegh MH, Carpenter CB: Tolerance and chronic rejection. Kidney Int 51(Suppl 58):S11–S14, 1997 Madsen JC, Yamada K, Allan JS, Choo JK, erhorn AE, Pins MR, Vesga L, Slisz JK, Sachs DH: Transplantation tolerance prevents cardiac allograft vasculopathy in major histocompatibility complex class I-disparate miniature swine. Transplantation 65:304– 313, 1998