Impact of maintenance immunosuppressive regimens--balance between graft protective suppression of immune functions and a near physiological immune response.

The Symphony study showed superior 1-year kidney graft outcome in patients on immunosuppression with tacrolimus/mycophenolate mofetil (Tacr/MMF). To analyze whether differences in clinical outcome between maintenance regimens may be explained by their impact on clinically relevant immune parameters, we assessed CD4 helper activity, immunoglobulin-secreting cell (ISC) formation, neopterin, sCD30, and intracellular cytokine production in a prospective study in 77 renal transplant recipients treated with cyclosporine A/azathioprine (CsA/Aza), CsA/MMF, Tacr/Aza or Tacr/MMF at 2 years post-transplant. Tacr- compared with CsA-based immunosuppression was independently associated with increased IL-2 (P<0.0001, CD4 cells; P=0.014, CD8 cells) and CD4 cell IL-4 responses (P=0.046; stepwise logistic regression) resulting in physiological responses in Tacr/Aza patients as compared with 25 healthy controls. MMF versus Aza treatment was proven to be an independent variable associated with suppression of CD4 cell IL-10 responses (P=0.008), B-cell IL-6R expression (P<0.0001) and ISC formation [P=0.020, staphylococcus cowan strain I (SAC I); P=0.021, pokeweed mitogen (PWM)]. Our data suggest that Tacr/MMF had the most effective impact on graft protective Th2 responses (enhanced CD4 cell IL-4 by Tacr, decreased CD4 cell IL-10 responses by MMF) and suppression of B-cell functions (MMF), whereas Tacr/Aza was associated with physiological IL-2 and IL-4 and stronger humoral responses which may reduce the risk of infectious disease complications.

Association of high IFN-gamma plasma levels with low B-cell counts in renal transplant recipients with stable long-term graft function.

Recently, we reported that patients with long-term stable good graft function had higher interferon-gamma (IFN-gamma) and lower IL-4 plasma levels late as compared with early post-transplant. These patients had more often detectable CD3(+)CD4(+)CD25(+)IFN-gamma(+)Foxp3(+) peripheral blood lymphocytes (PBL) late post-transplant than patients with impaired graft function. We therefore speculated that high plasma IFN-gamma late post-transplant might contribute to the maintenance of graft acceptance. Using ELISA and four-color flow cytometry, plasma cytokines and PBL subpopulations were measured in 65 renal transplant recipients with stable graft function late post-transplant. High IFN-gamma plasma levels were associated with low CD19(+) B PBL (r = -0.329; p = 0.009) and low activated CD3(+)CD8(+)DR(+) T PBL (r = -0.266; p = 0.035). Plasma IFN-gamma increased with time post-transplant (r = 0.288; p = 0.022) and was not associated with the dose of immunosuppressive drugs (p = n.s.). High plasma IFN-gamma was not associated with serum creatinine (r = 0.038; p = 0.765). Five patients showed evidence of chronic allograft nephropathy in previous biopsies and none of them exhibited increased plasma IFN-gamma. In patients with good long-term graft function, high IFN-gamma plasma levels were associated with low numbers of B PBL and activated CD8(+) T PBL. High IFN-gamma plasma levels might prevent the development of an immunological alloresponse and thereby contribute to the maintenance of graft acceptance.

Activation of Ca2+ -activated potassium channels is involved in lysophosphatidylcholine-induced monocyte adhesion to endothelial cells.

OBJECTIVE: Ca(2+)-activated K(+)-channels (BK(Ca)) play an important role in lysophosphatidylcholine (LPC)-induced endothelial dysfunction. Aim of our study was to investigate whether LPC-induced activation of BK(Ca) is also involved in monocyte adhesion to endothelial cells (EC). METHODS AND RESULTS: Measurement of membrane potential (MP) was performed using the fluorescence dye DiBAC. Adhesion of the monocytotic cell line U937 to EC was analysed by (3)[H]-thymidine-adhesion-assay. Expression of ICAM-1 and VCAM-1 were analyzed by FACS. LPC induced a hyperpolarization of EC in a dose-dependent manner with the maximum seen with 2 microM. This was prevented by the BK(Ca)-inhibitor iberiotoxin (IBX, 100nM). Adhesion of U937 cells to EC was increased after stimulation of EC with LPC. This effect was time-dependent with the maximum seen after 4h. LPC-induced adhesion was significantly reduced when EC were co-incubated with IBX, or NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI, 5 microM) and also blocked by addition of 2-aminoethoxydiphenylborate (2-APB, 100 microM) or the calcium-chelator BAPTA (10 microM). Stimulation of U937 cells with LPC did not result in an increased adhesion to unstimulated EC. CONCLUSION: Activation of the endothelial BK(Ca) plays an important role in monocyte adhesion to endothelial cells.

ATG induction in renal transplant recipients: Long-term hazard of severe infection is associated with long-term functional T cell impairment but not the ATG-induced CD4 cell decline.

BACKGROUND AND METHODS: We showed previously that rabbit ATG induction induces a strong decrease of CD4+ T cells together with impaired in vitro IL-2 secretion up to 1 year post-transplant. To further characterize long-term immunological effects of ATG induction 2 and 5 years post-transplant, we used sensitive intracellular cytokine analysis in the same prospective study of 84 renal transplant recipients (ATG, n=44). RESULTS: A significantly increased frequency of severe infectious disease (HR=2.0, p=0.027) as well as suppressed T cell functions were found within 2 years after ATG induction but not beyond (logistic regression (logreg): CD4 cell IL-10 responses, p=0.064; T cell proliferation, p=0.038). Impaired T cell proliferation at 2 years was associated with occurrence of severe infection (p=0.017). Importantly, a strong and persistent decrease of CD4 cell counts (p<0.0005 at 5 years) was independently associated with ATG induction (logreg p=0.002) but not related to functional CD4 cell impairment (helper activity/cytokine production) or an increased risk of infection. CONCLUSIONS: Severe infection up to 2 years after ATG induction was associated with impaired T cell proliferative capacity but not with the profound decline in CD4 cell counts that occurred after ATG induction and persisted up to 5 years.

Increased pretransplantation plasma kynurenine levels do not protect from but predict acute kidney allograft rejection.

Indoleamine 2,3-dioxygenase (IDO), an enzyme expressed in many cell types, catalyses degradation of tryptophan (Trp) to kynurenine (Kyn) and may exert immunosuppressive functions, mediated mainly by kynurenines. Therefore, increased Kyn concentrations would be expected to protect allografts from rejection. We conducted this study to examine whether Kyn has predictive value for kidney graft outcome. End-stage renal disease patients (n = 210) demonstrated an increased Kyn/Trp ratio compared with healthy controls (n = 30). Both Kyn and Trp levels were significantly higher in patients who subsequently developed acute rejection than in patients who did not (p < 0.001 and p < 0.001, respectively). Furthermore, pretransplantation Kyn and Trp plasma concentrations were significantly different in patients who went on to develop acute rejection (high values) or acute tubular necrosis (low values) (p = 0.007 and p = 0.021, respectively). After transplantation Kyn levels decreased. Approximately 3 days before biopsy-confirmed rejection, Kyn was significantly increased in patients with rejection compared with those without rejection (p < 0.001). Contrary to expectation, high Kyn plasma levels before transplantation were not predictive of low rejection risk. Although informative in overall terms, at the present stage, Kyn levels do not allow the concise risk differentiation of individual patients.

Association of pretransplant soluble glycoprotein 130 (sgp130) plasma levels and posttransplant acute tubular necrosis in renal transplant recipients.

OBJECTIVE: Previously, we reported that high pretransplant sIL-6R plasma levels are associated with posttransplant acute tubular necrosis (ATN). In this study, we examined associations of pretransplant plasma levels of sgp130 with ATN. PATIENTS AND METHODS: Pretransplant serum creatinine (Cr), plasma neopterin, and sgp130 levels were studied in 105 first renal transplant recipients who received grafts from deceased donors. Although 57 patients had immediate and sustained graft function, ATN was diagnosed in 30 patients within the first 11.3+/-7.8 posttransplant days and acute rejection in 18 patients during the first 27.1+/-27.6 days. RESULTS: Pretransplant serum Cr and plasma neopterin were similar in the three patient groups. Pretransplant sgp130 plasma levels, however, were significantly lower in patients with ATN than in patients with immediate graft function (P=0.004) or acute rejection (P=0.009). Multivariable logistic regression analysis for ATN showed an odds ratio of 4.3 of patients with pretransplant sgp130 less than or equal to 250 pg/mL with posttransplant ATN (P=0.006). CONCLUSION: Patients at risk of ATN showed immediately before transplantation low anti-inflammatory sgp130, suggesting a contribution of the IL-6 cytokine family to the development of ATN. It might be useful to measure IL-6 family plasma levels pretransplant to identify patients who are at an increased risk of developing ATN.

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+ blood lymphocytes in kidney transplant recipients with good long-term graft outcome.

There is evidence that interferon-gamma (IFN-gamma)-dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28(-) (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T- and B-cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of < or =1.8 mg/dl and 32 recipients with a serum creatinine of > or =2.0 mg/dl more than 100 days post-transplant. Cell subsets were measured in whole blood using four-color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN-gamma+ PBL than patients with good graft function (P = 0.017). In patients with good graft function, CD3+CD4+CD25+IFN-gamma+ PBL were associated with high Foxp3+, IL-2+, IL-12+, IL-4+, and IL-10+ CD3+CD4+CD25+ T PBL (P < 0.001), low CD3+CD8+CD28(-)Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage(-)HLA-DR+CD11c+CD123(-) DC1 (P = 0.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co-expression of IFN-gamma and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN-gamma+ cells in a subgroup of transplant recipients with good graft acceptance.

Fatty acids differentially influence phosphatidylinositol 3-kinase signal transduction in endothelial cells: impact on adhesion and apoptosis.

In contrast to n-6 fatty acids like arachidonic acid (AA), the anti-inflammatory potential of n-3 fatty acids such as docosahexaenoic acid (DHA) has been demonstrated. We examined the phosphatidylinositol (PI)3-kinase dependent effects of AA versus DHA on monocyte rolling, adhesion and transmigration through inflammatory activated human umbilical venous endothelial cells (HUVEC) as well as on apoptosis, to investigate the impact on vascular inflammation. HUVEC were pre-incubated with AA, DHA or sham, and stimulated with VEGF, TNF-alpha or staurosporine. Rolling and adhesion were investigated by means of a parallel flow chamber; transmigration was performed in a static assay. Activation of PI3-kinase was measured as phosphorylation of protein kinase B (Akt). Apoptosis was determined by caspase-3 activity and annexin-V analysis. Pre-incubation of HUVEC with DHA markedly decreased TNF-alpha-induced monocyte rolling, adhesion, and transmigration, although expression of endothelial adhesion molecules was unchanged. In contrast, AA increased TNF-alpha-induced rolling. Both fatty acids did not alter TNF-alpha-mediated upregulation of the adhesion molecules ICAM-1, VCAM-1, and E-selectin. The divergent effects of AA and DHA were abrogated with PI3-kinase inhibitors. After pre-incubation with DHA, VEGF-, TNF-alpha- and staurosporine-induced phosphorylation of Akt was decreased when compared to AA. DHA pre-incubation significantly increased staurosporine-induced apoptosis. In addition, DHA in comparison to AA augmented staurosporine-mediated increase in caspase-3 activity. In conclusion, DHA-induced a reduction in rolling, adhesion and transmigration of monocytes through inflammatory activated HUVEC that is in part PI3-kinase dependent. PI3-kinase driven phosphorylation of Akt and apoptosis of HUVEC as contribution to the resolution of inflammation is differentially modulated by DHA versus AA.

Evaluation of T-cell receptor repertoires in patients with long-term renal allograft survival.

The mechanisms underlying long-term acceptance of kidney allografts in humans under minimal or no maintenance immunosuppression are poorly understood. We analyzed the T-cell receptor (TCR) repertoires in circulating T cells of patients with long-term (> or = 9 years) renal allograft survival with (LTS-IS) and without immunosuppression (LTS-NoIS). T cells of LTS patients exhibited strongly altered TCR Vss usage, including an increased frequency of oligoclonality and a decreased frequency of polyclonality. All 3 LTS-NoIS and 12 of 16 LTS-IS patients demonstrated oligoclonality in at least three or more TCR V beta families, and the frequency of oligoclonality in these patients was significantly higher as compared to patients with well-functioning grafts at 3 years (p < 0.005 both), an uncomplicated course during the first year (p < 0.0001, both), acute rejection (p < 0.0001, both), chronic allograft nephropathy at 7 (p < 0.0001, both) or 13 years (p < 0.0001, both), dialysis patients (p < 0.0001, both) or healthy controls (p < 0.0001, both). In contrast to LTS patients, all other studied patient groups exhibited a polyclonal TCR repertoire. Our data indicate that TCR alteration is a common feature of long-term allograft outcome, which might be explained by clonal deletion, exhaustion of alloreactive T cells or predominant expression of particular T-cell subpopulations, such as regulatory T cells.

ATG induction therapy: long-term effects on Th1 but not on Th2 responses.

Antithymocyte globulin (ATG) induction therapy is associated with an increased long-term risk of infection- and cancer-related death. To analyze long-term effects of ATG induction on lymphocyte function, we prospectively assessed CD4 helper function, B-cell/monocyte and cytokine responses in 84 renal transplant recipients (ATG, n = 44) up to 1 year post-transplant. A PWM-driven allogeneic coculture system was used to assess helper function of CD4+ T cells and T-cell-dependent B-cell responses. SAC I was used for T-cell-independent stimulation of B-cell cultures. In vitro cytokine secretion and serum soluble CD30 (sCD30) were determined by enzyme-linked immunosorbent assay (ELISA). ATG induced a persistent decrease of peripheral blood lymphocyte counts compared with non-ATG treatment because of a predominant decrease of CD4+ T cells (4 months, 1 year; P < 0.0005) which was associated with a decreased CD28 expression (1 year, P = 0.02) and CD4 cell interleukin 2 (IL-2) response (4 months, P < 0.0005). However, Th2 responses (CD4 help, CD4 cell IL-4 and IL-10 responses, sCD30), which proved to be predictive of graft outcome, were not affected, and neither was the secretion of the lymphoma growth factors IL-6 and IL-10 by B cells and monocytes. Our data show that ATG induction therapy in immunological high-risk patients induces a profound long-term decrease in cell counts and Th1 but not Th2 responses of CD4+ T cells which may explain long-term effects on infection and post-transplant lymphoproliferative disease (PTLD) incidence because of inadequate T-cell control.