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Marshall, Sara E.1 2; McLaren, Andrew J.; Haldar, Neil A.; Bunce, Mike; Morris, Peter J.; Welsh, Ken I.

Clinical Transplantation

Background. Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. As cytokines are major regulators of the immune system, genetic variation in cytokine production or activity may influence susceptibility to acute rejection. This study sought to determine the impact of recipient cytokine and cytokine receptor polymorphisms on acute rejection after renal transplantation.

Methods. A total of 209 cadaveric renal transplant recipients were selected for analysis according to the presence or absence of graft rejection in the first 30 days after transplantation. DNA was genotyped for 22 polymorphisms in 11 cytokine and cytokine receptor genes using the polymerase chain reaction with sequence specific primers. Results were stratified by incidence and severity of rejection, and by HLA-DR mismatching.

Results. No association between any polymorphism and the incidence or severity of acute rejection was detected. In particular, no association was seen with tumor necrosis factor or interleukin-10 genotype, either alone or in combination.

Conclusions. We have failed to demonstrate any association between recipient cytokine genotype and acute rejection after cadaveric renal transplantation. Although more extensive studies may disprove these findings, it would seem premature to use recipient cytokine genotyping to predict transplant outcome, or to guide immunosuppressive therapy after transplantation.

Oxford Transplant Centre and Nuffield Department of Surgery, Oxford Radcliffe Hospitals, Oxford, United Kingdom

Received 16 February 2000.

Accepted 24 May 2000.

2 Address correspondence to: Dr. Sara E. Marshall, Oxford Transplant Centre, Churchill Hospital, Oxford OX3 7LJ, United Kingdom.

1 Supported by a Clinician Scientist Fellowship from the Medical Research Council.

The outcome of an immune response to any foreign antigen is influenced by the local microenvironment in which the encounter occurs, and this microenvironment is in part determined by cytokines (1). These small, locally-acting proteins interact to regulate immune responses through a highly intricate biological network. The classification of lymphocyte responses into “type 1” and “type 2,” according to the cytokines produced (2, 3), has provided an important conceptual framework within which to consider immune responses, and unraveling the complexity of cytokine interactions and their role in directing immune responses has become a major focus of immunological research. A large number of common single nucleotide polymorphisms of cytokine and cytokine receptor genes have been described (4). Although the function of many of these polymorphisms is unknown, evidence is accumulating that some influence gene function, for example, by altering the amino acid sequence of the protein (e.g., interleukin [IL]-4 receptor [5]) or by modifying transcriptional activity of the gene (e.g., tumor necrosis factor [TNF] [6], IL-6 [7], IL-10 [8]). Consequently, there has been considerable interest in studying these polymorphisms in a wide variety of inflammatory and immune-mediated diseases (9, 10).

Despite major advances in immunosuppressive therapy, acute allograft rejection remains a major cause of morbidity and mortality after solid organ transplantation (11, 12). Acute rejection occurs in 53% of cadaveric renal transplants in our center (13), and, although a major risk factor is donor-recipient disparity at the HLA-DR locus (14), additional factors are also involved. Genetic variability in cytokine production, which results in a shift of the cytokine milieu, could impact significantly upon the outcome of the immune response, and thus on the incidence and severity of acute rejection of an allograft.

Cytokine genotypes have previously been studied in patients undergoing solid organ transplantation, and certain polymorphisms have been implicated in the development of complications such as acute rejection and chronic allograft failure (15), although the role of individual gene variants is controversial. The most extensively analyzed are polymorphisms in the TNF and IL-10 genes, which have been shown to be associated with acute rejection in some (16, 17) but not all (18–21) studies. The situation is further complicated by evidence that the impact of specific polymorphisms varies in different transplant settings, with the combination of TNF −308A and IL-10 −1082A alleles being associated with increased acute rejection in heart transplantation (16), but decreased acute rejection in renal transplantation (17).

Perhaps such complexity is not surprising, given the intricacy of the cytokine network. The function of these biological proteins in humans has been difficult to unravel, with many cytokines having pleomorphic and even apparently contradictory effects (22). Furthermore, cytokines do not act in isolation but regulate both themselves and each other, and thus the net cytokine milieu is the product of many interacting proteins. As a result, analysis of a limited number of polymorphisms may be excessively reductionist, as these may fail to truly reflect the local microenvironment in which an immune response occurs.

We have previously described a unified polymerase chain reaction/sequence-specific primer (PCR-SSP)-based assay that permits the rapid genotyping of a wide variety of polymorphic immunoregulatory genes (24). This approach allows the integrated analysis of a large number of genetically distinct but functionally related polymorphisms, and is particularly suited to analysis of the intricate and pleomorphic cytokine network. We have further developed this assay and have applied it to the study of the role of genetic polymorphisms in cytokine and cytokine receptor genes in acute rejection after cadaveric renal transplantation.

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Patient selection.

This study was approved by the Central Oxfordshire Research and Ethics Committee. A total of 209 Caucasoid cadaveric renal transplant recipients from the Oxford Transplant Centre were selected for retrospective analysis according to the presence (n=114) or absence (n=95) of an acute rejection episode in the first 30 days after renal transplantation. Acute rejection was defined using clinical, biochemical, and histological criteria including, in all cases, a rise in creatinine >15% above baseline and characteristic histological features of rejection in a biopsy of the transplanted kidney. Steroid-responsive acute rejection (n=66) was said to have occurred if an individual experienced an episode of acute rejection within 30 days of transplantation with a serum creatinine that returned to baseline after a course of methylprednisolone (usually 500 mg daily for 3 days). If the creatinine failed to fall or return to baseline, patients underwent an additional biopsy to confirm the diagnosis of continuing acute rejection, and were defined as having steroid-resistant rejection (n=48). Patients included in the “no acute rejection” group were also selected by specific criteria, namely the absence of a >15% rise in creatinine in the first 30 days after transplantation, and no evidence of tubulitis, vasculitis, or a lymphocytic infiltrate on protocol biopsy (day 7 and day 28). No individual was highly sensitized (panel reactive antibodies >85% [IgG]) at the time of transplantation. All recipients were treated with cyclosporine, azathioprine, and prednisolone triple therapy after transplantation, and none received induction therapy with anti-lymphocyte agents.

Rejection status was classified by a single author (A.McL.), and all genotyping was performed without knowledge of the rejection status of each individual.

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Genotyping for cytokine polymorphisms.

All individuals were genotyped for 22 polymorphisms in 11 cytokine and cytokine receptor genes, namely, IL-1α (−889t/c [25]), IL-1β (−511t/c and +3962 t/c [26]), IL-1 receptor (Pst I 970c/t [27]), IL-1 receptor antagonist (Msp I 11100t/c [28]), IL-4 (−590t/c [29]), IL-4 receptor (+1902g/a [5]), IL-6 (−174g/c [7] and +3247g/a [24]), IL-10 (−1082a/g, −819c/t, −592c/a [8]), TNF (−308a/g, −238a/g, +488a/g [30], lymphotoxin (+249a/g, +365c/g, +720c/a [30]) and transforming growth factor-β1 (−880g/a, −509c/t, aa10L/P, and aa25R/P [31]). For ease of discussion, these polymorphisms are abbreviated to “recipient cytokine genotype” in this report, although it is recognized that many other polymorphisms in these and other genes may also contribute to the cytokine network.

Cytokine genotyping was performed using a unified PCR-SSP system (polymerase chain reaction with sequence-specific primers), where all assays are performed under identical amplification and detection conditions (24). The protocols for DNA extraction, PCR amplification, and gel electrophoresis have been described previously (24 and these assays have been reported previously (24, 30, 33–35). Assays for TNF, lymphotoxin, and IL-10 utilize both forward and reverse allele-specific primers, so that cis combinations of alleles can be directly amplified. Previous studies have demonstrated that, using this method, haplotypes can be inferred using a limited number of primer combinations caused by linkage disequilibrium (30).

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Study design and statistical analysis.

Phenotype, genotype, and allele frequencies were measured for all polymorphisms. Phenotype frequencies were obtained by counting the number of individuals in a population positive for an allele; allele frequencies were obtained by directly counting the number of chromosomes bearing an allele. Associations were assessed using 2 × n contingency table analysis and the chi-square test, with Yates’ correction or Fisher’s exact tests where appropriate. Odds ratio and the odds ratio 95% confidence interval were also calculated for significant associations.

To correct probability values for multiple comparisons, we applied a “two-set” approach (36), where frequencies of individual alleles and haplotypes were analyzed in a predetermined first set of 111 recipients, and then repeated in the second set of 98 recipients. Only those associations found to be associated (P <0.05) with rejection in both cohorts were considered to be significant, and the data were merged.

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A total of 209 renal transplant recipients were assessed for 22 polymorphisms in 11 cytokine and cytokine receptor genes. Genotype, phenotype, and allele frequencies are presented in Table 1. Extensive analysis revealed no differences in the distribution of the polymorphisms studied between patients who experienced acute rejection in the first 30 days after renal transplantation and those who did not. An increase in IL4 receptor 1902a/g heterozygotes in the acute rejection group was noted, but, as this association was present only in the first set of patients, this failed to achieve the criteria for significance for this study (data not shown). No other differences in genotype, allele, or phenotype frequency were detected.

Table 1

Table 1

Table 1A

Table 1A

{tabft}a Data for IL-4 receptor genotype are missing from three individuals because of lack of DNA.

b TGF, transforming growth factor.

Results were further analyzed according to whether recipients received an HLA-DR-matched or -mismatched allograft. As expected, HLA-DR mismatches were strongly associated with acute rejection (P <0.000002, data not shown), but further analysis of cytokine genotype failed to reveal any additional risk factors in either HLA-DR-matched or -mismatched groups.

Stratification was also performed according to rejection severity (i.e., steroid-responsive or steroid-resistant rejection). Small differences were noted when individuals with steroid-resistant rejection were compared with individuals who had no rejection, such as weak associations of polymorphisms in the IL-4 receptor (P <0.03) and the IL-1β promoter (P <0.03, data not shown). However, once again when individuals were divided into first and second sets, these failed to achieve statistical significance as defined for this study. In contrast, HLA-DR mismatching was strongly associated with steroid-resistant rejection in both sets, as well as in the overall group.

Considerable attention has focused on the role of polymorphisms in TNF and in IL-10 in acute rejection after solid organ transplantation. The combination of TNF −308A/IL-10 −1082A has been associated with a decreased number of acute rejection episodes after heart transplantation (16), but increased incidence and severity of acute rejection after renal transplantation (17). Thus, when analyzing the data presented here, particular attention was taken with regard to these polymorphisms. However, no significant associations were detected, and no evidence for synergy between these variants was demonstrated (Table 2).

Table 2

Table 2

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The immune system is regulated by an array of cytokines, which influence cellular activation, differentiation, and function. A large number of common polymorphisms in cytokine and cytokine receptor genes have been identified, igniting interest in the role of these subtle variants in the regulation of cytokine production and function. It is clear from in vitro studies that some of these polymorphisms have functional significance, although their precise effect may be controversial (6, 37, 38). Furthermore, in vivo population studies suggest that certain polymorphisms may influence susceptibility to infectious, allergic, and autoimmune disorders (10).

In this study, we analyzed a large number of functionally related cytokine and cytokine receptor polymorphisms in an attempt to unravel the impact of these genetic variations on acute rejection after renal transplantation. This was facilitated by the development of a unified PCR-SSP assay system that permitted the simultaneous analysis of 22 polymorphisms in 11 cytokine and cytokine receptor genes (24). Although this is an attractive method, there is a high probability of type I statistical errors as the analysis of such a large number of variations may result in apparent associations simply by chance. Indeed, the majority of association studies are never replicated, as they are easily compromised by inappropriate controls and inadequate statistical methods (39). This may be addressed by applying a Bonferroni correction factor, where, in essence, the statistical P-value is multiplied by the number of analyses performed (40), but this approach may result in relevant but minor associations being overlooked (41). As it was anticipated that the impact of any single polymorphism would be small, we chose instead to apply a “two-set” approach (36). Using this method, a polymorphism was required to be associated with a P-value <0.05 in both the overall group and the two independent subgroups for it to be considered significant. By ensuring that any association would have to be replicated in a similar but independent patient group to meet the criteria for significance, we have attempted to avoid identifying spurious associations. Although it is possible that the use of this approach has led us to ignore an important association, we have adopted it as it guarantees that any association described is sufficiently robust to be replicated at least once.

Another potential confounding factor was population stratification. To address this, we endeavored to ensure that our patient group was as homogenous as possible: all individuals were of Caucasoid ethnic origin, all received the same initial immunosuppression, none were highly sensitized at the time of transplantation, and all diagnoses of rejection were confirmed by biopsy of the transplanted kidney.

Results were analyzed according to the presence or absence of acute rejection, and were further stratified by severity of rejection and by HLA-DR mismatching. The latter was included, as not only is HLA-DR mismatching a major determinant of acute rejection (14), it has also been suggested that the impact of cytokine genotype may be influenced by the degree of immune activation initiated by an MHC class II mismatch (17). No association between any cytokine polymorphism and acute graft rejection was seen. In particular, we found no evidence for a role for either TNF or IL-10 polymorphisms, either individually or synergistically.

These negative findings contrast with the work of others, and a number of reasons for this may be postulated. First, this study addressed the role of recipient cytokine genotype in cadaveric renal transplantation, although much of the previously published work has concerned heart (16, 18), lung (31), or liver (19) transplantation. As the heart and the lung are considered to be more immunogenic than the kidney, it is conceivable that the impact of some of these polymorphisms is only manifest when the immune system is maximally activated. Alternatively, the immunosuppressive regimen used may influence the production and function of different cytokines: patients undergoing heart or lung transplantation usually receive anti-lymphocyte agents at the time of transplantation, and this may affect the impact of genetic variation in cytokine production. Interestingly, in the largest previous study in renal transplantation, the majority of patients were treated with cyclosporine monotherapy (17), whereas, in the study described here, all patients were on cyclosporine, azathioprine, and prednisolone triple immunosuppression. Thus, it is possible that differences in immunosuppressive therapy may enhance or negate the importance of cytokine genotype.

A further feature of this study was that the presence or absence of acute rejection was defined by stringent clinical and histological criteria. Individuals thus chosen probably represent the extremes of the immune response to a transplanted organ, and although it is theoretically possible that, by excluding individuals who did not meet these criteria, we have missed a critical subgroup, this is unlikely: the policy of this transplant center is to monitor all individuals with plasma creatinine levels a minimum of four times weekly in the first 30 days after transplantation, and to biopsy any individual with a rise in creatinine of >15% above baseline, as well as to perform protocol biopsies at day 7 and day 28. Thus, it seems improbable that a significant subgroup of individuals with active rejection were excluded.

Nonetheless, differences in patient selection and disease definition may well underlie the variability of results seen in different transplant cohorts: in this study, all acute rejection episodes were confirmed histologically, whereas, in the largest previous study of cytokine polymorphisms in renal transplantation, biopsies were obtained in less than 50% of cases (17). In addition, the nature of the groups being compared may differ: in the latter study, groups were analyzed according to the presence or absence of two or more rejection episodes (17), whereas, in the study described here, individuals with no acute rejection episodes in the first 30 days after transplantation were compared with those who experienced acute rejection in the same time period. These subtle differences may well explain the variation in results from different centers.

Thus, our failure to identify an association between recipient cytokine genotype and acute rejection does not necessarily indicate that these polymorphisms do not play a role in renal transplantation. More extensive studies of larger patient groups, or of additional polymorphisms and combinations of polymorphisms, may yet reveal important associations. In addition, as cytokines derived from the graft itself also contribute to the cytokine microenvironment, donor cytokine genotype may influence the rejection response. Indeed, in a simultaneous study of donor cytokine polymorphisms in renal transplantation, we have identified a significant association between donor IL-6 genotype and incidence and severity of acute rejection, although the mechanism by which polymorphisms in IL-6 influence transplant outcome is unclear. 3 Analysis of donor-recipient genotype combinations may also provide useful information, although, in view of the extensive statistical analysis required, such studies will probably require large multicenter patient cohorts.

In summary, we have found no evidence to suggest that recipient cytokine genotype as defined by 22 cytokine polymorphisms determines the incidence or severity of acute rejection after cadaveric renal transplantation. Although more extensive studies may disprove these findings, we would suggest that it is premature to use recipient cytokine genotyping to predict transplant outcome, or to guide immunosuppressive therapy after transplantation.

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3 Marshall SE et al. Transplantation 2000; in press.
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