Chronic Rejection: Insights from a Novel Immunosuppressive-Free Model of Kidney Transplantation
Martin Gasser*,
Ana Maria Waaga-Gasser*,
Joana E. Kist-van Holthe,
Xueli Yuan,
Susanne M. Lenhard,
Kald A. Abdallah and
Anil Chandraker
Laboratory of Immunogenetics and Transplantation, Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.
Correspondence to Dr. Anil Chandraker, Laboratory of Immunogenetics and Transplantation, Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115. Phone: 617-732-7412; Fax: 617-732-5254; E-mail: achandraker{at}rics.bwh.harvard.edu
ABSTRACT. The use of immunosuppressive drugs in models of chronicrejection may limit their usefulness for mechanistic studies.We have developed a new minor histocompatibility-mismatchedrat kidney transplant model without the need for immunosuppression.Kidneys from LEW (RT1l) donors were transplanted to congenicWF.1L (RT1l) recipients and compared with the reversed straincombination and isogenic controls. Urinary protein excretionwas measured serially in all recipients; kidneys were harvested90, 120, and 180 d after transplantation for morphologic analysisand cytokine gene expression. In vitro lymphocytic reactivityand cytokine analysis of mixed lymphocyte reaction (MLR) culturesupernatants by ELISA was also carried out. LEW into WF.1L kidneygrafts developed proteinuria starting 120 d after transplantationand were associated with morphologic changes of focal segmentalglomerulosclerosis together with interstitial cell infiltrates,upregulated gene expression of IL-1, IL-2, and TNF-/-, as wellas IL-2, IFN-, and TNF- production by lymphocytes in MLR culturesupernatants. WF.1L kidneys transplanted into LEW recipientsdid not develop chronic rejection and had upregulation of Th2cytokines, both within the allograft and in MLR supernatantof recipient lymphocytes cultured with WF.1L cells. Furthermore,these lymphocytes produced both Th1 and Th2 cytokines when culturedwith WF cells, unlike lymphocytes from the LEW isografts, whichproduced Th1 cytokines when challenged with WF cells. Thesestudies show that indirect allorecognition can cause strain-dependentchronic rejection associated with Th1-like cytokine production,whereas production of Th2 cytokines is associated with protectionfrom the development of chronic rejection.
The term chronic rejection as applied to kidney transplantswas first used by Harlan et al. to describe histologic changesin kidney allografts that occurred later and were differentfrom those seen in acute rejection (1). An immunological basiswas assumed to be the cause of the lesions seen; hence, theterm chronic rejection was used. It is now well recognized thatnonimmunological factors can exacerbate the development of thischronic alloimmune response. A newer term, chronic allograftnephropathy, is now commonly used to emphasize the multifactorialnature of the damage seen in the transplanted kidneys. The termchronic rejection is now usually reserved for purely immunologicallymediated injury (2). There is still no doubt that an alloimmuneresponse lies at the heart of most, if not all, chronic allograftnephropathy. The nature of this chronic alloimmune responseis also believed to be different from that of acute rejection;therefore, it is important to develop good models of chronicrejection to understand this form of injury and how best totreat it (3).
Although all solid organs develop some form of chronic alloimmune-mediatedinjury, the exact nature of this injury varies from organ toorgan and the degree of injury even varies in different organstransplanted across the same strain combination (4–6).Hence, insights gained from cardiac or other solid organ transplantmodels may not be wholly relevant to the development of chronicrejection in kidney allografts.
The most commonly used chronic rejection model for kidney transplantationis the rat F334-to-LEW transplant model (5,7,8). Although thishas provided many insights into the development of chronic rejection,it suffers from one major drawback: it requires the use of ashort course of cyclosporine to modify the acute rejection andprevent a high attrition rate among the transplanted animals.Cyclosporine itself has been shown to contribute to the developmentof chronic allograft nephropathy; therefore, its use in a modelcomplicates mechanistic studies into the development of chronicrejection, not only because of its immunosuppressive effectsbut also because of its nephrotoxic effects (9). In addition,calcineurin inhibitors are thought to interfere with the inductionof transplantation tolerance (10,11).
To address these concerns, we decided to develop a new modelof chronic kidney rejection in the rat that does not requirethe use of immunosuppressive drugs. Guttmann et al. have shownthat LEW recipients of MHC-matched congenic WF.1L (RT1l) cardiactissue develop chronic cardiac rejection beginning 60 d aftertransplantation and becoming full blown by 120 d (12–14).In this study, we examined whether this minor histocompatibility-mismatchedmodel may be suitable for the analysis of chronic kidney rejectionin an otherwise untreated animal. WF.1L and LEW kidneys weretransplanted into LEW and WF.1L recipients, respectively, andmorphologic and functional changes were compared with syngeneiccontrols.
Animals and Operative Technique
Inbred male rats, 8 to 10 wk of age and weighing 200 to 250g, were used throughout the experiments. Pathogen-free LEW (RT1l)rats were purchased from Harlan Sprague Dawley (Indianapolis,IN). The congenic WF.1L (LEW)/GUT (RT1l) rats were a generousgift from Dr. Ronald Guttmann (McGill University, Montreal,Canada). The WF.1L rats were originally derived from backcrossingLEW to WF (RT1u), and the haplotype was fixed by selecting l/Lprogeny at F6. Animals were bred by brother/sister mating formore than 20 generations before being shipped to our laboratory(14). All animal experimentation described was conducted inaccord with the National Institutes of Health Guidelines forthe Care and Use of Laboratory Animals. Our laboratory has beenbreeding WF.1L animals for the past 3 yr.
The kidneys were flushed with 3 ml of cold lactated Ringer’ssolution before removal and stored transiently in the same coldsolution (4°C) before engraftment. They were transplantedheterotopically with a short aortic segment anastomosed end-to-sideto the abdominal aorta of the recipient, and the renal veinwas joined in a similar manner to the inferior vena cava. Theureter was divided, and the proximal third was anastomosed end-to-endto the recipient ureter. All anastomoses were performed with10-0 Prolene (Ethicon, Somerville, NJ). The left kidney wasthen removed. The time between the release of the vascular clampsand the return of obvious uniform cortical blood flow was approximately35 to 45 s. The contralateral right native kidney was removed10 d later, at which time the graft had recovered from any dysfunctionresulting from transient ischemia and the integrity of the ureteralanastomosis in the transplant could be examined.
Experimental Groups
Kidneys were transplanted from LEW donors into WF.1L recipients(LEW into WF.1L) as well as in the reversed strain combination(WF.1L into LEW). LEW into LEW and WF.1L into WF.1L isograftsserved as controls for both experimental groups, and no immunosuppressivedrugs were given to any of the groups.
Renal Function
All animals had monthly 24-h urine collections between day 30and day 180 (n = 5/group). Protein excretion was determinedby measuring turbidity after precipitation of urinary proteinand interaction with 3% sulfosalicylic acid (Fisher Scientific,Fair Lawn, NJ). Turbidity was assessed by absorbance at a wavelengthof 595 nm using a UV-1201 spectrophotometer (Shimadzu, Kyoto,Japan) (7).
Histology
Graft morphology was examined at 90, 120, and 180 d. Representativeportions of all allografts and isografts were fixed in 10% bufferedformalin at the time of removal for histomorphological examination.Paraffin sections were stained with hematoxylin and eosin, periodicacid–Schiff, Mason’s trichrome for collagen, andVerhoeff’s elastin for elastic fibers and evaluated bylight microscopy.
Two examiners blinded to the group from which the sections cameexamined matched trichrome-stained sections independently. Asemiquantitative scoring system for fibrosis was used, withspecimens graded from 0 to 3, where 0, 1, 2, and 3 indicatedminimal, mild, moderate, and severe fibrosis, respectively.A similar scale was used to score the degree of interstitialmononuclear cell infiltration.
Portions of the kidney grafts harvested at 180 d after transplantationwere also snap frozen and stored at -80°C for the determinationof cytokines and growth factors using an RNase protection assay.
Mixed Lymphocyte Reaction
Donor-strain lymph nodes or splenocytes were harvested, andmononuclear cells were separated by passing the tissue througha 60-gauge sterile stainless steel sieve. The stimulating cells(3 x 105 per well) were then irradiated (3000 rads) and coculturedwith responder T cells (3 x 105 per well) in 96-well flat-bottomplates (Costar, Cambridge, MA) containing RPMI 1640 medium (BioWhittaker,Walkersville, MD), 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin,2 x 10-5 mol/L 2-mercaptoethanol, and 5 mmol/L Hepes. The plateswere incubated at 37°C with 5% CO2 for 96 h. Proliferationwas assayed by measuring the DNA incorporation of [3H]thymidine(New England Nuclear-Dupont, Boston, MA), added for the final6 h of culture, using a -scintillation counter (Beta Plate;Wallac, Gaithersburg, MD). All experiments were done in quadruplicate,and the results are expressed as cpm (experimental minus background)±SEM. Background cultures were set up with responder cells alone.
ELISA for Rat TNF-, IFN-, IL-2, IL-4, and IL-10
From the mixed lymphocyte reaction (MLR) culture, supernatantswere harvested after 48 h of incubation at 37°C with 5%CO2 to determine TNF-, IFN-, IL-2, IL-4, and IL-10 production.Culture supernatants were assayed by ELISA using BioSource CytoscreenRat ELISA Kits (BioSource, Camarillo, CA).
RNase Protection Assay
RNase protection was performed using the Riboquant Multi-ProbeRNase Protection assay system (PharMingen, San Diego, CA). RNAwas isolated from kidney grafts using Trizol. 32P-labeled probeswere synthesized from the rCK-1 Multi-Probe Template Set (PharMingen)and were hybridized overnight with RNA samples in hybridizationbuffer according to the manufacturer’s instructions. Sampleswere digested with RNase and T1 mix in RNase buffer, and protectedprobes were purified and run on a 5% acrylamide gel in 0.5%TBE buffer. Kidney graft control RNA and a dilution of the probeset (serving as size markers) was run in parallel. The gel wasabsorbed onto filter paper, dried, and exposed on Kodak photographicpaper at -70°C for 24 h. The RNA was analyzed by a phosphorimagerusing ImageQuant software (Molecular Dynamics, Sunnyvale, CA),allowing accurate quantification of mRNA.
Statistical Analyses
The results are expressed as arithmetic means ± SEM.The MLR data are presented as differences between the proliferativeresponses of the tested cells minus the proliferative responsesof the responder and stimulator cells tested alone (cpm). Statisticalsignificance among groups was ascertained using the t test andthe Mann-Whitney test. Urine protein excretion was comparedamong all groups using the Kruskal-Wallis test for several independentsamples and the Mann-Whitney test for two independent samples.The differences were considered to be significant at P <0.05.
Survival and Renal Function
All recipients in the four groups survived the 180-d follow-upperiod. Proteinuria was determined sequentially for all recipientsin all groups (Figure 1). LEW kidneys transplanted into LEWrecipients showed no evidence of increased urinary protein excretionby day 180 after transplantation (9.6 ± 5 mg). WF.1Lkidneys transplanted into LEW recipients developed mild proteinuria(16.7 ± 6.0 mg), but not significantly different fromWF.1L isografts (15.26 ± 4.9 mg). By contrast, WF.1Lrecipients of LEW kidneys developed progressive proteinuriaevident by day 60 and becoming significantly different fromthe other groups by day 120 (28.3 ± 6.1 mg at day 180;P < 0.0001)
Figure 1. Progressive increase in proteinuria occurred in animals in group 1 versus group 3 and 4 controls. Increasing proteinuria was associated with signs of chronic rejection in group 1 animals. Data are presented as means ± SEM (n = 8/group per time point; P < 0.0001).
Histology
Graft morphology from each of the groups harvested at day 180after transplantation mirrored the proteinuria data. Transplantedkidneys from the two isogenic control groups were essentiallynormal when examined 180 d after transplantation (Table 1).Allografts from the LEW-into-WF.1L group showed histologic changesof chronic rejection, with persistent focal cellular interstitialinfiltration and focal glomerulosclerosis (Figure 2 and Table 1).Kidneys from the reverse strain combination, WF.1L kidneystransplanted into LEW recipients, showed very minor morphologicchanges, with only mild glomerulosclerosis and occasional cellularinfiltrates (Figure 2). Arteriosclerotic alterations of corticalsmall vessels or interstitial fibrosis were not significantlydifferent when compared with both isograft groups.
Table 1. Semiquantitative scoring for fibrosis and interstitial mononuclear cell infiltrate in transplanted kidneys harvested 180 d after transplantation (n = 6 per group)
Figure 2. Histology from group 1 and group 3 transplanted kidneys at 24 wk after transplantation. (A and C) Group 3 isografts. (B, D, and E) Group 1 isografts. There is evidence of mononuclear cell infiltration, fibrosis, and focal segmental glomerulosclerosis in the group 1 isografts compared with the group 3 isografts, which appear essentially normal at 24 wk after transplantation (trichrome staining). Magnification, x150.
Mixed Lymphocyte Reaction
To investigate whether the differences seen in the developmentof chronic rejection in the LEW-into-WF.1L and WF.1L-into-LEWtransplants was attributable to recipient immunoresponsiveness,we tested the proliferative response of lymphocytes harvestedfrom each group to irradiated antigen-presenting cell (APC)from naïve LEW, WF.1L, and WF animals.
As expected, the primary MLR response of splenic and lymph node(LN) lymphocytes from naïve WF.1L and LEW animals to irradiatedminor histocompatibility-mismatched LEW or WF.1L APC was minimal(splenic cells, 1237 ± 186 versus 1523 ± 201,respectively; Figure 3C). Recipient splenic and LN lymphocytesfrom the LEW animals primed with a WF.1L kidney (which developedchronic rejection) showed a significant response to naïveirradiated APC of donor origin (Figure 3A). Interestingly, acomparable response was observed in recipient lymphocytes tonaïve irradiated APC of donor origin from the reverse straincombination, which did not develop chronic rejection.
Figure 3. Recipient splenic and LN lymphocytes (WF.1L) from group 1 (LEW into WF.1L) showed a significant response to naïve irradiated LEW antigen-presenting cell (APC) (A). A comparable response was observed in the reversed strain combination in group 2 (WF.1L into LEW) compared with the response of recipient lymphocytes (LEW) to naïve irradiated WF.1L APC (B). The proliferation was higher when WF.1L (group 1) or LEW (group 2) recipient lymphocytes were cultured with naïve irradiated WF APC (A and B). The response of splenic and LN lymphocytes from naïve WF.1L and LEW animals to irradiated LEW and WF.1L APC was minimal (C). Data are presented in cpm ± SEM (n = 4).
The proliferative response of recipient lymphocytes from bothallografted groups was higher when cultured with naïveirradiated APC from WF compared with WF.1L (splenic cells group1, 8045 ± 1456 cpm, group 2, 5998 ± 721 cpm; Figure 3, A and B).However, the proliferation of lymphocytes fromLEW allografted animals to WF APC was significantly lower thanthe response of lymphocytes taken from LEW isografted animals(splenic cells, 15,937 ± 2284 [not shown] versus 5998± 721; Figure 3), indicating that prior transplantationof LEW animals with a WF.1L organ blunts the immunoresponsivenessof LEW lymphocytes to WF APC.
Enzyme-Linked Immunosorbent Assay
Culture supernatants obtained from the MLR assays were examinedfor cytokine production by ELISA. Lymphocytes from animals thatdeveloped chronic rejection (LEW into WF.1L) had a Th1 phenotype,with production of TNF-, IFN-, and IL-2 but not IL-4 and IL-10,when stimulated with cells mismatched for minor or major histocompatibilityantigens (Figure 4A). Even though LEW recipients of WF.1L kidneysmounted a significant alloimmune response in the MLR-to-WF.1LAPC, the supernatant indicated a Th2 response, with productionof IL-4 and IL-10 rather than Th1 cytokines. LEW animals previouslytransplanted with LEW isografts produced a strong Th1 responsein terms of IFN- and IL-2 but small amounts of IL-4 and IL-10when stimulated with WF cells. Priming LEW animals with a WF.1Lallograft, before stimulating with WF APC, changed the cytokineresponse to a mixed Th1/Th2 response, with increased productionof TNF-, IFN-, IL-2, IL-4, and IL-10. Thus, it is possible thatthe Th2 response (presumably against the minor histocompatibilityantigens) in these animals regulates the more aggressive Th1response to WF MHC.
Figure 4. Cytokine analysis of the culture supernatants obtained from mixed lymphocyte reaction assays. Group 1 animals showed TNF-, IFN-, and IL-2 but barely detectable IL-4 and IL-10 production (A) when tested against minor and major mismatch antigens. In contrast, the pattern was reversed in group 2 animals (B), with greater production of IL-2, IL-4, and IL-10 observed in culture supernatants of the group 2 splenic lymphocytes. (C and D) Cytokine production in mixed lymphocyte reaction supernatants from groups 3 and 4, respectively. Data are presented as means ± SEM (n = 4; P < 0.0001).
Molecular Changes
To confirm that the cytokine expression was not merely an invitro phenomenon, we examined cytokine gene expression in allograftsharvested at regular intervals by RNase protection assay formRNA levels of IL-1 and -, IL-2, -3, -4, -5, -6, and -10, TNF-and -, and IFN- and compared the results with those from naïveorgans. These data mirrored the cytokine expression found inthe culture supernatants. At 90 and 180 d after kidney transplantation,IL-1, IL-2, and TNF- and - gene expression were upregulatedin grafts from the LEW-into-WF.1L group compared with thosefrom the WF.1L-into-LEW and LEW-into-LEW groups (Figure 5).By contrast, IL-4 was upregulated in the WF.1L kidneys transplantedinto LEW recipients.
Figure 5. Cytokine gene expression in kidney allografts of groups 1 to 4. (A) By 90 d after kidney transplantation, IL-1, IL-2, and TNF- and - gene expression was significantly upregulated in grafts from LEW into WF.1L (group 1) compared with those from WF.1L into LEW (group 2) and the syngeneic controls LEW into LEW (group 3) and WF.1L into WF.1L (group 4). In contrast, IL-4 gene expression was only upregulated in group 2. IL-5 and IL-6 were detectable but not significantly different from those in the control grafts. Data are presented as cytokine/GAPDH ratio (n = 4/group). (B) Differences in cytokine gene expression were still present at 180 d after kidney transplantation.
For more than two decades, the F344-to-LEW model of kidney transplantationhas been the most popular model of chronic renal allograft rejection(5). The F344 (RT1lvr) rat has a variant l haplotype that differsfrom LEW (RT1l) in the class 1 E/C region but is identical toLEW in the immunodominant RT1.A and RT1.B regions (15). In thismodel, cyclosporine is usually given for 10 d to overcome acuteallograft rejection. Renal allografts in this strain combinationsurvive for a prolonged period and develop marked functionalimpairment and histologic changes compatible with chronic rejection/chronicallograft nephropathy. As cyclosporine is also known to be nephrotoxic,the use of cyclosporine makes this model less than ideal. Severalcongenic models have been used to analyze the role of the majorand minor histocompatibility antigen differences in the rejectionprocess, mainly for differentiation between isolated MHC loci(e.g., the RT.1A and RT.1B/D complex), responsible for the inductionof the immune response (16–19). It is now clear that theexperimental process that initially defined the "major" histocompatibilitycomplex similarly defined the "minor" loci by the slower rejectioncaused by their antigens. The combination of several minor histocompatibilitydiscrepancies may bring about rejection as rapidly as a wholeMHC difference (20,21). Recently, a heterotopic cardiac allograftmodel in the WF.1L (LEW)-to-LEW inbred rat strain combinationwas identified as an experimental model for systematic studiesof chronic vascular rejection (12,14). RT1Al serotyping andanalysis of the LEW-type TNF- polymorphism by PCR methods haveshown that this congenic line has the LEW immunodominant classI MHC as well as the MHC class II loci RT.1B and RT.1Dof l haplotype.In this MHC-identical combination, indefinite survival (>100d) of the cardiac allografts occurred in the absence of recipientimmunosuppression. The grafts revealed morphologic featuresof progressive chronic vascular rejection that were highly reproducibleas a function of time. Extensive infiltration by T cells andmacrophages within the vessel wall were variably associatedwith minimal nonocclusive intimal accumulation of actin andmyointimal cells or with changes of occlusive or subocclusiveintimal thickening associated with minimal vascular wall infiltrationby macrophages. Advanced vasculopathy was already present 60d after transplantation.
To study chronic renal allograft rejection based on this cardiactransplant model, we developed a new MHC-matched, but minorhistocompatibility-mismatched, rat kidney model, the LEW-into-WF.1Lmodel. This new model develops the functional and histologicfeatures of chronic renal allograft rejection without the needof calcineurin inhibitor-based immunosuppression. Although avariable degree of focal interstitial mononuclear cell infiltrationwas seen in the kidney allografts compared with the cardiacallograft model mentioned above, significant vascular changes,even after 180 d, were not detectable in either the LEW-into-WF.1Lor the WF.1L-into-LEW kidney transplant model. Differences inthe way that kidney and cardiac transplants respond to immunosuppressive/immunoregulatoryregimens in the same model are well documented (6). Vasculopathyis often not a dominant finding in kidney allografts undergoingchronic rejection compared with cardiac allografts. When itdoes occur, it appears to be associated with sensitization andlate acute rejection (22), neither of which occurs in this model.The lack of vascular changes in the kidney model may be attributableto differences in the relative importance of the direct/indirectpathway of antigen recognition, the type of antigen-processingcell, the route of antigen processing, and so forth.
WF.1L recipients of LEW kidneys developed increased urinaryprotein excretion and grade 1 to 2 lesions of chronic rejectionaccording to the Banff classification 180 d after transplantation(23). By contrast, LEW recipients of WF.1L kidneys developedlittle chronic rejection. Strain-dependent development of chronicrejection has been documented in the F334-to-LEW combination(24). In this model, F344 kidneys transplanted into LEW recipientsdevelop chronic rejection, and reversing the strain combinationdoes not result in the development of chronic rejection. Thereasons for this are not totally apparent but are likely attributableto the combination of antigen processing and the repertoireof T cells available to recognize foreign antigens. Minor alloantigensrequire APC processing before activating a T cell response andhence are dependent on the indirect pathway of allosensitization(25,26). Most described studies of allograft rejection basedon minor histocompatibility-mismatched strains have used hearttransplant models either in the rat or in the mouse (27–29).Kidney grafts from the LEW-to-WF.1L congenic stain combination(group 1) revealed a slow but significant process of chronicrejection, with typical signs of glomerulosclerosis and representativecytokines, i.e., TNF-, IFN-, IL-1, and IL-2, associated withchronic rejection after 90 d, before the development of significantproteinuria. A similar pattern of cytokine expression was seenin grafts from this group harvested 180 d after transplantation,implying an association between this cytokine profile and thedevelopment of chronic rejection. Cells obtained from theseanimals demonstrated alloreactivity in vitro not only againstmajor but also against minor mismatch antigens that may reflectthe in vivo ongoing slow rejection process. Although the overallproliferative responses were reduced compared with those ofcells obtained from naïve controls, this observation couldbe explained by the fact that the alloreactive T cell clonesize is relatively small in a minor mismatched combination andby the effect of age on the vigor of the T cell response (28).In the supernatants of these cultures, TNF-, IFN-, and IL-2were detectable in this strain combination (LEW into WF.1L).Interestingly, in the reversed strain combination, in whichno significant signs of chronic rejection were observed, IL-4and IL-10 were detectable not only in the culture supernatantsbut also in the kidney grafts of those animals, reflecting type2 cytokine expression, previously associated with transplantationtolerance (30). It has been speculated that Th2 responses maybe responsible for the development of chronic rejection (31);in contrast, we found that chronic rejection was associatedwith a Th1 response, in keeping with human studies that haveshown that alloreactive T cells cloned from chronically rejectingpatients were universally of the Th1 phenotype (32).
Antigen-dependent risk factors for the development of chronicrejection, i.e., major and minor mismatch antigens, have beenobserved to have their origins in the activation of T cellsthrough the immunologically mediated recognition of allograftantigens. Our observations indicate that the outcome of theallograft response in a given host, rejection or tolerance (ora mixed response), may also be dependent on whether a particularindividual develops a Th1 or a Th2 response to the mismatchedantigen. Although transplantation of MHC-mismatched WF kidneysinto LEW recipients leads to an aggressive form of acute rejection,and lymphocytes from the LEW isografts produced a predominantlyTh1 response to WF cells, WF.1L kidneys transplanted into LEWrecipients did not develop chronic rejection (33). LEW lymphocytesfrom these animals produced predominantly Th2 cytokines whenchallenged with WF.1L cells and a mixed Th1/Th2 response whenchallenged with WF cells.
These studies reiterate the complexity of the alloimmune responsein the development of chronic rejection and highlight the importanceof donor-recipient pairing in the development of this form ofinjury.
Acknowledgments
This work was supported by National Institutes of Health grantAI02888. We thank Dr. Ron Guttmann for generously providingus with breeding pairs of the WF.1L (LEW)/GUT (RT1l) [Guttmann]rats.
Footnotes
*Co-first authors.
Dr. Barbara Murphy served as Guest Editor and supervised thereview and final disposition of this manuscript.
Harlan WR Jr, Holden KR, Williams GM, Hume DM: Proteinuria and nephrotic syndrome associated with chronic rejection of kidney transplants. N Engl J Med 277: 769–776, 1967
Halloran PF: Call for revolution: A new approach to describing allograft deterioration. Am J Transplant 2: 195–200, 2002[CrossRef][Medline]
Womer KL, Vella JP, Sayegh MH: Chronic allograft dysfunction: Mechanisms and new approaches to therapy. Semin Nephrol 20: 126–147, 2000[Medline]
Diamond JR, Tilney NL, Frye J, Ding G, McElroy J, Pesek-Diamond I, Yang H: Progressive albuminuria and glomerulosclerosis in a rat model of chronic renal allograft rejection. Transplantation 54: 710–716, 1992[Medline]
Schaub M, Stadlbauer TH, Chandraker A, Vella JP, Turka LA, Sayegh MH: Comparative strategies to induce long-term graft acceptance in fully allogeneic renal versus cardiac allograft models by CD28-B7 T cell costimulatory blockade: Role of thymus and spleen. J Am Soc Nephrol 9: 891–898, 1998[Abstract]
Azuma H, Chandraker A, Nadeau K, Hancock WW, Carpenter CB, Tilney NL, Sayegh MH: Blockade of T-cell costimulation prevents development of experimental chronic renal allograft rejection. Proc Natl Acad Sci U S A 93: 12439–12444, 1996[Abstract/Free Full Text]
Chandraker A, Azuma H, Nadeau K, Carpenter CB, Tilney NL, Hancock WW, Sayegh MH: Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection. J Clin Invest 101: 2309–2318, 1998[Medline]
Remuzzi G, Perico N: Cyclosporine-induced renal dysfunction in experimental animals and humans. Kidney Int Suppl 52: S70–S74, 1995[Medline]
Chandraker A, Russell ME, Glysing-Jensen T, Willett TA, Sayegh MH: T-cell costimulatory blockade in experimental chronic cardiac allograft rejection: Effects of cyclosporine and donor antigen. Transplantation 63: 1053–1058, 1997[CrossRef][Medline]
Smiley ST, Csizmadia V, Gao W, Turka LA, Hancock WW: Differential effects of cyclosporine A, methylprednisolone, mycophenolate, and rapamycin on CD154 induction and requirement for NFB: Implications for tolerance induction. Transplantation 70: 415–419, 2000[CrossRef][Medline]
Guttmann RD, Forbes RD, Zheng S, Busque S: Cyclosporine and the reversibility of chronic vascular rejection. Transplant Proc 26: 2564–2566, 1994[Medline]
Forbes RD, Zheng SX, Gomersall M, Guttmann RD: Irreversible chronic vascular rejection occurs only after development of advanced allograft vasculopathy: A comparative study of a rat cardiac allograft model using a retransplantation protocol. Transplantation 63: 743–749, 1997[CrossRef][Medline]
Forbes RD, Zheng SX, Gomersall M, al-Saffar M, Guttmann RD: Evidence that recipient CD8+ T cell depletion does not alter development of chronic vascular rejection in a rat heart allograft model. Transplantation 57: 1238–1246, 1994[Medline]
Guttmann RD, Mahabir RN, Lindquist RR: Renal transplantation in the inbred rat. X. A model of "weak histocompatibility" by major locus matching. Transplantation 8: 369–378, 1969[Medline]
Quigley RL, Wood KJ, Morris PJ: The relative roles of major and minor histocompatibility antigens in the induction of immunologic unresponsiveness by blood transfusion. Transfusion 29: 789–793, 1989[CrossRef][Medline]
Cramer DV, Qian SQ, Harnaha J, Chapman FA, Estes LW, Starzl TE, Makowka L: Cardiac transplantation in the rat. I. The effect of histocompatibility differences on graft arteriosclerosis. Transplantation 47: 414–419, 1989[Medline]
Hutchinson IV, Morris PJ: The role of major and minor histocompatibility antigens in active enhancement of rat kidney allograft survival by blood transfusion. Transplantation 41: 166–170, 1986[Medline]
Zimmermann FA, Davies HS, Knoll PP, Gokel JM, Schmidt T: Orthotopic liver allografts in the rat: The influence of strain combination on the fate of the graft. Transplantation 37: 406–410, 1984[Medline]
Graff RJ, Bailey DW: The non-H-2 histocompatibility loci and their antigens. Transplant Rev 15: 26–49, 1973[Medline]
Simpson E: Non-H-2 histocompatibility antigens: Can they be retroviral products? Immunol Today 8: 176–177, 1987[CrossRef]
Sijpkens YW, Doxiadis II, van Kemenade FJ, Zwinderman AH, de Fijter JW, Claas FH, Bruijn JA, Paul LC: Chronic rejection with or without transplant vasculopathy. Clin Transplant 17: 163–170, 2003[CrossRef][Medline]
Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, Croker BP, Demetris AJ, Drachenberg CB, Fogo AB, Furness P, Gaber LW, Gibson IW, Glotz D, Goldberg JC, Grande J, Halloran PF, Hansen HE, Hartley B, Hayry PJ, Hill CM, Hoffman EO, Hunsicker LG, Lindblad AS, Yamaguchi Y, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55: 713–723, 1999[CrossRef][Medline]
de Heer E, Davidoff A, van der Wal A, van Geest M, Paul LC: Chronic renal allograft rejection in the rat: Transplantation-induced antibodies against basement membrane antigens. Lab Invest 70: 494–502, 1994[Medline]
Sano Y, Ksander BR, Streilein JW: Murine orthotopic corneal transplantation in high-risk eyes: Rejection is dictated primarily by weak rather than strong alloantigens. Invest Ophthalmol Vis Sci 38: 1130–1138, 1997[Abstract/Free Full Text]
Benichou G, Valujskikh A, Heeger PS: Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice. J Immunol 162: 352–358, 1999[Abstract/Free Full Text]
Izutani H, Miyagawa S, Shirakura R, Matsumiya G, Nakata S, Shimazaki Y, Matsuda H: Evidence that graft coronary arteriosclerosis begins in the early phase after transplantation and progresses without chronic immunoreaction: Histopathological analysis using a retransplantation model. Transplantation 60: 1073–1079, 1995[Medline]
Kishimoto K, Sandner S, Imitola J, Sho M, Li Y, Langmuir PB, Rothstein DM, Strom TB, Turka LA, Sayegh MH: Th1 cytokines, programmed cell death, and alloreactive T cell clone size in transplant tolerance. J Clin Invest 109: 1471–1479, 2002[CrossRef][Medline]
Youssef AR, Otley C, Mathieson PW, Smith RM: Effector mechanisms in murine allograft rejection: Comparison of skin and heart grafts in fully allogeneic and minor histocompatibility antigen-mismatched strain combinations. Transpl Int 15: 302–309, 2002[CrossRef][Medline]
Waaga AM, Gasser M, Kist-van Holthe JE, Najafian N, Muller A, Vella JP, Womer KL, Chandraker A, Khoury SJ, Sayegh MH: Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo. J Clin Invest 107: 909–916, 2001[Medline]
Shirwan H: Chronic allograft rejection: Do the Th2 cells preferentially induced by indirect alloantigen recognition play a dominant role? Transplantation 68: 715–726, 1999[CrossRef][Medline]
Kist-van Holthe JE, Gasser M, Womer K, Najafian N, Dong V, Samsonov DV, Geehan CS, Chandraker A, Sayegh MH, Waaga AM: Regulatory functions of alloreactive Th2 clones in human renal transplant recipients. Kidney Int 62: 627–631, 2002[CrossRef][Medline]
Akalin E, Chandraker A, Russell ME, Turka LA, Hancock WW, Sayegh MH: CD28-B7 T cell costimulatory blockade by CTLA4Ig in the rat renal allograft model: Inhibition of cell-mediated and humoral immune responses in vivo. Transplantation 62: 1942–1945, 1996[CrossRef][Medline]
Received for publication June 17, 2003.
Accepted for publication November 22, 2003.
Top
Abstract
Introduction
, IL-2, and TNF-
/-
, and TNF-
cpm (experimental minus background)± SEM. Background cultures were set up with responder cells alone. 
B: Implications for tolerance induction. Transplantation 70: 415–419, 2000[CrossRef][Medline]
