Growing evidence suggests that the chemokine CXCL10 may be particularly important for the immune response to transplant. Indeed, in knockout models of the CXCL10 gene or of its receptor gene, called CXC chemokine receptor 3 (CXCR3), cardiac transplant undergoes permanent engraftment (1–3). Furthermore, neutralization of CXCL10 with monoclonal antibodies prolongs the allograft survival in both cardiac and small-bowel allograft rejection (3,4). Of particular importance, the intragraft expression of CXCL10 has been reported in association with clinical rejection of acute renal (5), lung (6), and cardiac (7,8) allografts. Thus, CXCL10-CXCR3 interactions appear to play an important role in the pathogenesis of graft failure caused by rejection in multiorgan models. Recent evidence indicates that CXCR3 and CXCL10 are also highly expressed in conjunction with the development of transplant vasculopathy in cardiac allografts (9) and that glomerular infiltration by CXCR3+ T cells is associated with transplant glomerulopathy (10).
In the present study, we investigated CXCL10 expression and distribution in tissue specimens obtained from patients suffering from acute rejection (AR) or chronic allograft nephropathy (CAN). In AR, CXCL10 was expressed by infiltrating monocytes/macrophages, whereas CAN was characterized by wide CXCL10 expression not only at level of infiltrating inflammatory cells but also of vascular, tubular, and glomerular structures. Furthermore, as assessed on 316 cadaveric kidney recipients, pretransplant quantitation of CXCL10 serum levels were able to predict the recipient’s risk of severe AR, CAN, and transplant failure. The comparison with a number of variables that have all been proposed as possible predictors of transplant failure, including soluble CD30 (sCD30) (11), showed that high pretransplant serum CXCL10 levels have a high predictive power in identification of subjects with high risk of allograft failure, a finding that might be used for the individualization of immunosuppressive therapies.
MATERIALS AND METHODS
A total number of 316 cadaveric kidney transplants were carried out at the Florence Transplant Center between January 1991 and June 2001, had pretransplant sera available, and were tested retrospectively for CXCL10 and sCD30 content levels. All patients were on hemodialysis before transplant and underwent measurements of main hematochemical parameters every 6 months. Samples were collected on the morning before dialysis. An aliquot was obtained from the serum sample that was used for final cytotoxic cross-match performed within 1 month before the date of transplantation. The other aliquot was randomly selected from all available samples obtained from the same patients at least 6 months before. In addition, 48 age- and sex-matched controls were tested. All the recipients were white. The demographic characteristics of the recipients were the followings: age 46.33±6.4 years; sex M/F 209/107; dialytic age 38.3±2.1 months; and original disease glomerulopathies 43.3%, ADPKD 21.5%, interstitial nephritis 12%, nephroangiosclerosis 11.5%, and others 11.7%. Ninety-three percent of the patients received cyclosporine-based immunosuppression, whereas the remaining 7% received a FK-based immunosuppression. Only one patient had more than one transplant. Human leukocyte antigen (HLA) typings and panel reactive lymphocytotoxic antibody (PRA) determinations were performed at the Tissue Typing Laboratory of Azienda Ospedaliera Careggi of Florence, Italy. Patients with 5% or greater antibody reactivity against a randomly chosen lymphocyte test panel were categorized as sensitized and represented 13% of all subjects. HLA-A+B+DR mismatches were three or less in 44% and three or more in 56% of patients. Donor age was 45.5±0.93 years, and cold ischemia time was 20.2±0.36 hours. Information on graft function and patient survival was documented at 1, 3, and 6 months and at 1, 2, 3, 4, and 5 years. Death with a functioning graft was not counted as graft failure in this analysis. The median follow-up time after transplant was 39 months. Delayed graft function was defined as the need of at least one session of dialysis during the first 7 days after transplantation (12). The procedures used in this study were in accordance with the Regional Ethical Committee on human experimentation.
Renal-biopsy specimens from a total number of 22 subjects were used throughout the study. Biopsy specimens were obtained from 10 patients suffering from AR and from 12 patients affected by CAN. Only biopsy proved AR were used for statistical analysis. AR episodes were classified according to Banff classification (13) and treated with steroids. AR episodes showing vascular components at biopsy (grade II or III) and failure to reverse after a course of steroids were treated with rabbit anti-human thymocyte globulin (ATG).
Immunohistochemical staining was performed as previously described (14). For double label immunohistochemistry, the anti-CXCL10 (Peprotech, Rocky Hill, NJ) pAb was applied first, and AEC was used as peroxidase substrate. Sections were subsequently exposed to anti-CD68 (EBM11; Dako, Glostrup, Denmark), anti-von Willebrand factor (vWf) (rabbit anti-human polyclonal antibody; Dako), anti-cytokeratin (CK, C-11; Sigma, Saint Louis, MO), and anti-α-smooth muscle actin (SMA; 1A4; Sigma), and Vector SG (Vector Laboratories, Burlingame, CA) was used as a chromogen. No counterstain was applied.
In Situ Hybridization
Cloning and sequencing of the CXCL10 probe and in situ hybridization were performed as previously described (14). For combined in situ hybridization and immunohistochemistry, after hybridization with the CXCL10 probe, RNAse digestion and appropriate washings, sections were stained with anti-vWf or anti-α-SMA Abs and then subjected to autoradiography, as reported earlier (14).
Serum CXCL10 (R&D Systems, Minneapolis, MN) and sCD130 levels were assayed by a quantitative sandwich immunoassay using commercially available kits, as described previously (11,15).
Statistical analysis was performed using SSPS software (SPSS, Inc., Evanston, IL). Because of nonparametric distribution, comparisons of serum CXCL10 and sCD30 levels among different groups were performed by Mann-Whitney U test for unpaired data. Correlation between two variables was ascertained by linear regression analysis and Spearman’s correlation test as appropriate. To test the independent effects of different variables on renal-allograft survival, multiple regression analysis was used, and partial correlation coefficients were computed. Kaplan-Meier estimates were used to generate an overall survival curve for transplanted grafts, and differences among groups were assessed by log-rank test. P<0.05 was considered statistically significant. Results are expressed in the text as mean±SEM unless otherwise stated.
CXCL10 is Highly Expressed in Kidney of Subjects with AR and CAN
CXCL10 mRNA appeared to be highly expressed in biopsy specimens from kidneys of patients affected by AR (Fig. 1, A to C), as assessed by in situ hybridization. In kidneys from patients with AR, positive cells were mainly identifiable as infiltrating inflammatory cells, which in many cases surrounded both vessels and tubular structures (Fig. 1, A and C). Sections hybridized with a sense CXCL10 RNA probe showed virtually no signal (Fig. 1B). Double immunostaining for CXCL10 and α-SMA, CK, vWf, and CD68 confirmed that in AR, monocytes/macrophages were the main source of this chemokine (Fig. 1C).
Biopsy specimens from kidneys of patients affected by CAN were also examined. High CXCL10 mRNA (Fig. 1, D to H) expression was consistently found even in these specimens, but the signal was not only localized to infiltrating inflammatory cells but also to proximal and distal tubules, as well as to medullary collecting ducts (Fig. 1, D and E). Moreover, some resident glomerular cells and vascular structures expressed CXCL10 mRNA (Fig. 1, F to H).
Combined in situ hybridization and immunohistochemistry or double immunostaining for CXCL10 and α-SMA, CK, vWf, and CD68 provided direct evidence that vascular smooth muscle cells (SMC) (Fig. 1H), in addition to endothelial cells, tubular epithelial cells, and monocytes/macrophages (data not shown), were the main sources of CXCL10.
CXCL10 Pretransplant Serum Levels Predict Recipient’s Risk of Early, Severe Acute Rejection, Chronic Allograft Nephropathy, and Graft Failure
Pretransplant serum levels of CXCL10 and sCD30 were assessed in 316 adult kidney-graft recipients by appropriate enzyme-linked immunoadsorbent assay methods. Kidney graft recipients had a significantly higher pretransplant serum CXCL10 content before transplantation than 48 adult healthy controls (138.0±7.0 vs. 80.4±4.8; P=0.0006 pg/mL) (Fig. 2A). To assess whether pretransplant serum CXCL10 levels in graft recipients were stable over time, 265 of 316 (84%) patients for whom an additional serum sample was available underwent a second CXCL10 serum assay. The time interval between the two serum collections was at least 6 months. Linear regression analysis demonstrated a strong relationship between the two assays (R2=0.793; P=0.000001), thus indicating that pretransplant serum CXCL10 levels are a stable parameter.
The assignment of all patients to two groups based on subsequent graft outcome revealed important differences. Indeed, patients with normally functioning grafts showed significantly lower pretransplant serum CXCL10 levels than patients who experienced graft failure (129.0±6.8 vs. 218.2±28.9 pg/mL; P=0.0007) (Fig. 2A). In agreement with previous results (11), assessment of sCD30 in the same samples demonstrated that patients with normally functioning grafts also exhibited lower levels than patients who experienced graft failure (114.0±3.1 vs. 155.5±31.8 pg/mL; P=0.04).
Accordingly, the 74 patients who underwent rejection episodes within the first month after transplantation showed higher pretransplant serum CXCL10 levels than nonrejectors (156.0±15.8 vs. 124.9±7.47 pg/mL; P=0.04) (Fig. 2B), and the 14 patients who required ATG treatment because of severe rejection, showing vascular components at biopsy (grade II or III) and failure to reverse after a course of steroids, were characterized by even higher pretransplant mean serum CXCL10 levels (277.14±65.8 pg/mL; P=0.004) (Fig. 2B). Interestingly, patients with very high CXCL10 levels (>200 pg/mL) also showed a significant inverse correlation between CXCL10 levels and day to first rejection (R2=0.24; P=0.048). Finally and more importantly, those patients who developed CAN also showed significantly increased pretransplant serum CXCL10 levels (193.2±36.9 pg/mL; P=0.03) (Fig. 2B). No differences were observed in serum pretransplant CXCL10 levels with regards to age, sex, year of transplantation, original disease, transplant number, type or duration of dialysis, PRA, or occurrence of delayed graft function (data not shown).
Predictive Values of Pretransplant Serum CXCL10 Levels and Other Factors for Graft Failure
Stratification of patients in three groups according to pretransplant serum CXCL10 levels (<100 pg/mL, n=163; 100–150 pg/mL, n=69; >150 pg/mL, n=84) and subsequent lifetime analysis (Kaplan-Meier and log-rank test) performed separately for the two extreme groups showed highly significant differences in 5-year survival rates (95.7% vs. 79.7%, P=0.0002 in total and P=0.00001 between groups 1 and 3) (Fig. 3). No influence of pretransplant serum CXCL10 on patient survival could be observed, and no differences for the length of surveillance period were found among groups showing different pretransplant CXCL10 serum levels (data not shown).
To estimate the relative risks for developing graft failure in relation to different parameters, multivariate Cox regression analysis was performed with graft failure as a dependent variable, whereas recipient age and sex, number of HLA-A, -B, and -DR mismatches, original disease, type of immunosuppression, PRA, number of previous transplant, donor age, occurrence of delayed graft function, and pretransplant serum levels of CXCL10 and sCD30 were considered as covariables. In particular, CXCL10 was entered as a dichotomous variable for values greater than 150 pg/mL and because previous studies demonstrated an increased risk of graft failure only when sCD30 levels are greater than 100 U/mL (14), sCD30 was analyzed as a categoric variable for values below and above 100 U/mL. The results confirmed a significant increased risk (risk ratio 2.801; confidence interval 1.201–6.536; P=0.017) for developing graft failure in such patients (Table 1). In agreement with previous results (12), multivariate analysis confirmed the importance of two other variables, donor age and delayed graft function, that had a significant predictive power for graft failure (Table 1).
Despite the administration of immunosuppressants, allograft rejection remains the primary cause of human renal-transplant failure. AR is particularly frequent in the early posttransplant period and despite the graft loss in only a small percentage of subjects represents a leading cause of morbility and hospitalization and predisposes renal allografts to the development of CAN (16). Large experimental evidence emphasizes the important role of CXCL10 in the initiation and amplification of host alloresponses that lead to AR. CXCL10 not only mediates leukocyte recruitment to rejecting allografts but is also a critical initiator of alloimmune response to the antigen (17); inasmuch, CXCL10 drives T-cell proliferation to allogenic and antigenic stimulation and interferon-γ secretion in response to antigenic challenge (17). Accordingly, CXCL10-deficient mice have impaired T-cell responses, impaired contact hypersensitivity response, limited inflammatory cell infiltrates, and are unable to control viral infections (17). In addition to its powerful effects within immune responses (17), CXCL10 also alters vascular cell functions (18–24). Indeed, CXCL10 can induce SMC (19) and mesangial cell proliferation (14,19,20) and also acts as a powerful angiostatic factor (21,22) on activated endothelium (22–24). The dual targets of CXCL10 suggest a possible role for this chemokine, not only in the pathogenesis of AR but also of CAN. Indeed, CAN is characterized by endothelial cell injury, vascular SMC proliferation, and mesangial expansion (1).
In this study, we demonstrate that CXCL10 is highly expressed not only in the kidney of subjects with AR but also with CAN. In AR, the main sources of CXCL10 were infiltrating inflammatory cells, particularly macrophages. More importantly, we demonstrate for the first time that the source of CXCL10 in CAN are several cell types, including not only infiltrating mononuclear cells but also vascular and tubular structures. These findings provide clear evidence that persistent CXCL10 expression can be found in CAN, and this observation provides a further possible link between AR and CAN. In a previous study, we suggested that pretransplant serum CXCL10 might predict the long-term success of renal transplantation (15). Furthermore, Hu et al. (25) found that CXCL10 is increased in the urine of patients with AR and is a more sensitive and predictive parameter than serum creatinine in terms of monitoring the response to antirejection therapy. On the basis of this experimental evidence, we also investigated whether pretransplant determination of CXCL10 serum levels may predict the recipient’s risk of severe, early AR and CAN.
It has been a primary goal of transplant immunologists to develop tests for assessing the pretransplant risk of patients for immunologic rejection and graft failure. Various risk factors— including recipient age and sex, number of HLA-A, -B, and -DR mismatches, original disease, type of immunosuppression, PRA, number of previous transplant, donor age, delayed graft function and pretransplant serum levels of sCD30 greater than 100 U/mL— have all been proposed as means for differentiating between patients with a good chance of long-term survival of a renal allograft and those with a poor chance. However, at least in our patients, only pretransplant serum CXCL10 levels greater than 150 pg/mL and the occurrence of delayed graft function had a predictive power of allograft loss. Accordingly, pretransplant quantitation of CXCL10 serum levels were also able to predict the recipient’s risk of early, severe AR and CAN. Interestingly, patients with very high CXCL10 levels also showed a significant inverse correlation between CXCL10 levels and day to first rejection, suggesting that the highest CXCL10 levels correspond with the least days for occurrence of AR.
Therefore, it is reasonable to speculate that patients with high serum CXCL10 pretransplant levels develop severe AR episodes because of increased immune activation but are also more prone to generation of intimal hyperplasia, chronic endothelial cell damage, and vascular necrosis that result in CAN and finally in graft failure (17–24). Finally, the demonstration that pretransplant serum CXCL10 levels are a clinically useful parameter for the identification of subjects exhibiting high risk of AR, CAN, and graft failure suggests that these subjects may require heavier posttransplant immunosuppressive regimens.
1. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol
2000; 18: 217.
2. Hancock WW, Gao W, Csizmadia V, et al. Donor-derived IP-10 initiates development of acute allograft rejection
. J Exp Med
2001; 193(8): 975.
3. Hancock WW, Lu B, Gao W, et al. Requirement of the chemokine receptor CXCR3
for acute allograft rejection
. J Exp Med
2000; 192(10): 1515.
4. Zhang Z, Kaptanoglu L, Haddad W, et al. Donor T cell activation initiates small bowel allograft rejection
through an IFN-γ-inducible protein-10-dependent mechanism. J Immunol
2002; 168(7): 3205.
5. Segerer S, Cui Y, Eitner F, et al. Expression of chemokines and chemokine receptors during human renal transplant rejection
. Am J Kidney Dis
2001; 37(3): 518.
6. Agostini C, Calabrese F, Rea F, et al. CXCR3
and its ligand CXCL10 are expressed by inflammatory cells infiltrating lung allografts and mediate chemotaxis of T cells at sites of rejection
. Am J Pathol
2001; 158(5): 1703.
7. Melter M, Exeni A, Reinders ME, et al. Expression of the chemokine receptor CXCR3
and its ligand IP-10 during human cardiac allograft rejection
2001; 104(21): 2558.
8. Fahmy NM, Yamani MH, Starling RC, et al. Chemokine and chemokine receptor gene expression indicates acute rejection
of human cardiac transplants. Transplantation
2003; 75(1): 72.
9. Zhao DX, Hu Y, Miller GG, et al. Differential expression of the IFN-inducible CXCR3
-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T Cell chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection
. J Immunol
2002; 169(3): 1556.
10. Akalin E, Dikman S, Murphy B, et al. Glomerular infiltration by CXCR3
+ ICOS+ activated T cells in chronic allograft nephropathy
with transplant glomerulopathy. Am J Transplant
2003; 3(9): 1116.
11. Süsal C, Pelzl S, Döhler B, et al., for the Collaborative Transplant Study. Identification of highly responsive kidney transplant recipients using pretransplant soluble CD30
. J Am Soc Nephrol
2002; 13: 1650.
12. Perico N, Cattaneo D, Sayegh MH, et al. Delayed graft function in kidney transplantation. Lancet
2004; 364: 1814.
13. Solez K, Benediktsson H, Cavallo T, et al. Report of the Third Banff Conference on Allograft Pathology (July 20–24, 1995) on classification and lesion scoring in renal allograft pathology. Transplant Proc
1996; 28: 441.
14. Romagnani P, Lazzeri E, Lasagni L, et al. IP-10 and Mig production by glomerular cells in human proliferative glomerulonephritis and regulation by nitric oxide. J Am Soc Nephrol
15. Rotondi M, Rosati A, Buonamano A, et al. High pretransplant serum levels of CXCL10/IP-10 are related to increased risk of renal allograft failure. Am J Transplant
2004; 4(9): 1466.
16. Kahan BD, Ponticelli C, eds. Principles and practice in renal transplantation. London, Martin Dunitz Ltd 2000, p. 846.
17. Dufour JH, Dziejman M, Liu MT, et al. IFN-gamma-inducible protein 10 (IP-10;CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol
2002; 168(7): 3195.
18. Lazzeri E, Romagnani P. CXCR3
-binding chemokines: multifaceted therapeutic targets. Curr Drug Targets Immune Endocr Metabol Disord
2005; 5: 109.
19. Wang X, Yue TL, Ohlstein EH, et al. Interferon-inducible protein-10 involves vascular smooth muscle cell migration, proliferation, and inflammatory response. J Biol Chem
1996; 271: 24286.
20. Romagnani P, Beltrame C, Annunziato F, et al. Role for interactions between IP-10/Mig and their receptor (CXCR3
) in proliferative glomerulonephritis. J Am Soc Nephrol
1999; 10: 2518.
21. Bonacchi A, Romagnani P, Romanelli RG, et al. Signal transduction by the chemokine receptor CXCR3
: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem
2001; 276: 9945.
22. Romagnani P, Annunziato F, Lasagni L, et al. Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J Clin Invest
2001; 107: 53.
23. Romagnani P, Lasagni L, Annunziato F, et al. CXC chemokines: the regulatory link between angiogenesis and inflammation. Trends Immunol
2004; 25(4): 201.
24. Lasagni L, Francalanci M, Annunziato F, et al. An alternatively spliced variant of CXCR3
mediates the IP-10, Mig and I-TAC induced-inhibition of endothelial cell growth and acts as functional receptor for PF-4. J Exp Med
2003; 197: 1537.
25. Hu H, Aizenstein BD, Puchalski A, et al. Elevation of CXCR3
-binding chemokines in urine indicates acute renal-allograft dysfunction. Am J Transplant
2004; 4(3): 432.