The pharmacodynamic effect of sirolimus: individual variation of cytokine mRNA expression profiles in human whole blood samples.

Sirolimus (SRL) has become an important alternative to calcineurin inhibitors due to its unique mechanism of action. Since rejection and poor graft outcome are still frequent problems despite therapeutic-range blood concentrations, pharmacodynamic measurements of its immunosuppressive effects would be of great clinical value to optimize treatment in individual patients. We performed a human whole blood assay using real time cytokine RT-PCR for the pharmacodynamic assessment of SRL. IL-2, IL-4 and IL-6 mRNA levels were quantitatively determined upon T-cell-specific stimulation in healthy individuals (n=11; in vitro) and in kidney-transplant patients (n=3; ex vivo). Furthermore, IL-2 protein secretion and T-cell proliferation was measured. After 24h incubation we observed a stronger suppression of IL-2 and IL-4 mRNA expression upon SRL addition (p<0.005; p<0.005) versus 4h (p<0.05; p<0.05). SRL effects displayed a remarkable interindividual variation, which proved to be independent of the concentration applied. Notably, 3/11 and 2/11 individuals had unaffected IL-2 and IL-4 mRNA expression after 4h incubation with SRL, respectively. In contrast, a general suppression of IL-2 protein secretion and T-cell proliferation was induced. Analysis of kidney-transplant patients verified interindividual variation and proved comparability of in vitro and ex vivo effects. We describe an individual degree of SRL-sensitivity that may correlate with clinical efficacy. Rather than analysis of one single peak, we suggest determination of two absolute cytokine mRNA peak levels for the pharmacodynamic assessment of SRL. However, prospective clinical studies are necessary to determine whether individual degrees of SRL-sensitivity correlate with clinical outcome.

Cooperative influence of the interleukin-6 promoter polymorphisms -597, -572 and -174 on long-term kidney allograft survival.

Recently, we demonstrated an association of the IL-6 promoter polymorphism at position -174 (G-->C) with kidney allograft survival whereby carriers of the -174GG genotype were identified as having superior graft survival. As two additional polymorphisms were discovered in the neighborhood at positions -572 (G-->C) and -597 (G-->A), respectively, and as functional studies revealed a cooperative impact of all three on the IL-6 gene transcription, we investigated whether there is a combined effect on kidney transplant outcome. We determined IL-6 promoter haplotypes -597 (G-->C)/-572 (G-->A)/-174 (G-->C)(-597/-572/-174haplotype) using a PCR system with sequence-specific primers in 158 patients after primary cadaveric kidney transplantation. We here show that the -597 and -174 polymorphism are in tight-linkage disequilibrium and that homozygous carriers of the GGG-597/-572/-174 haplotype (GGG/GGG genotype) have superior 3-year graft survival rates compared with the 8.0-fold increased risk of premature graft loss in all other patients. Interestingly, patients carrying the GGG/GCG genotype had the lowest allograft survival rate. Thus determination of the combined -597/-572/-174 genotype allows for further differentiation of -174GG patients into subgroups and consequently for a more accurate identification of patients at risk. Our results indicate that the three polymorphisms act in a cooperative fashion and we provide evidence for an exceptional clinical impact of the IL-6-597/-572/-174 genotype on the success of kidney transplantation.

Sensitivity of whole-blood T lymphocytes in individual patients to tacrolimus (FK 506): impact of interleukin-2 mRNA expression as surrogate measure of immunosuppressive effect.

BACKGROUND: To optimize immunosuppressive treatment in individual transplant patients, functional measurements of the effects of tacrolimus (FK 506) are of clinical importance. Previous investigations have demonstrated the occurrence of tacrolimus-resistant production of interleukin-2 (IL-2) in vitro, which may explain in part why rejection episodes are still a frequent problem despite attainment of therapeutic blood concentrations and HLA matching. However, an adequate surrogate marker to define the tacrolimus response in individual patients has not been established. METHODS: We investigated the immunosuppressive effects of tacrolimus on anti-CD3/anti-CD28 T-cell costimulation in a human whole-blood assay, analyzing T-cell proliferation, activation marker expression (CD25, CD69), IL-2 protein expression, and cytokine mRNA expression in vitro (n = 11 healthy individuals). We also quantified IL-2 mRNA expression in patients undergoing tacrolimus (n = 4) or cyclosporin A (CsA; n = 4) monotherapy before ex vivo living-donor kidney transplantation. RESULTS: T-cell proliferation; CD25, CD69, and IL-2 concentrations; and IL-4 mRNA were significantly decreased in vitro. In contrast, cytokine mRNA profiles revealed variable tacrolimus sensitivity. Whole-blood samples from 3 of 11 healthy individuals demonstrated marked suppression of IL-2 mRNA expression (>50%) when tacrolimus was administered in vitro. When CsA was added to whole-blood cultures, the influence on IL-2 mRNA expression was comparable to that of tacrolimus in 9 of 11 individuals. Two individuals responded conversely, indicating that differences in the in vitro response to tacrolimus and CsA among individuals may be attributable to potential heterogeneity in the involvement of the CD28 pathway. Kinetic profiles of IL-2 mRNA expression also revealed individually distinct degrees of calcineurin inhibitor sensitivity in patients undergoing tacrolimus or CsA monotherapy before living-donor kidney transplantation. CONCLUSIONS: Our results suggest an individual degree of calcineurin inhibitor sensitivity of activated whole-blood lymphocytes based on IL-2 mRNA expression. Our approach is potentially valuable for identifying transplant patients in whom IL-2 mRNA expression is unaffected or even enhanced after initiation of immunosuppressive therapy. Such individuals may be less sensitive to the immunosuppressive agent and therefore at increased risk of transplant rejection. Prospective studies are necessary to determine the correlation of IL-2 mRNA expression with the clinical risk of transplant rejection.