Hartmut Dietrich

Post‐Transplant sCD30 and Neopterin as Predictors of Chronic Allograft Nephropathy: Impact of Different Immunosuppressive Regimens

Immunological monitoring for chronic allograft nephropathy (CAN) is of great potential interest. We assessed serum soluble CD30 (sCD30) together with in vitro Th2‐type responses (IL‐4, IL‐10, CD4 helper activity) and neopterin in a prospective study of 84 renal transplant recipients with 2‐year follow‐up. Patients were randomized to CsA/Aza, CsA/MMF and Tacr/Aza, respectively, to analyze the effect of immunosuppression on posttransplant sCD30 and neopterin. ATG induction and acute rejections did not alter sCD30 levels whereas CMV disease was associated with transient upregulation of sCD30 (p = 0.003 at 4 months) and sustained upregulation of neopterin (corrected for graft function (Neo/CR) p = 0.005 at 2 years). Tacr versus CsA treatment proved to be an independent variable associated with downregulation of 1‐year sCD30, which was positively related to Neo/CR (p = 0.007 and 0.01, respectively; logistic regression). Importantly, increased 1‐year sCD30 and Neo/CR were associated with decreased glomerular filtration rate at 2 years (p = 0.02 and p < 0.0005, respectively) and evidence of CAN (p < 0.0005). High 1‐year sCD30 could not be attributed to enhanced Th2‐type responses and was not associated with HLA antibody formation. Our data suggest that elevated sCD30 and neopterin predict graft deterioration by CAN. Tacr effectively downregulates these responses and might be of advantage in patients with elevated sCD30 or neopterin.

Bone marrow transplantation demonstrates medullar origin of CD34+ fibrocytes and ameliorates hepatic fibrosis in Abcb4−/− mice

Bone marrow (BM)‐derived stem cells and CD34+ fibrocytes are associated with fibrogenesis in several organs. In an Abcb4−/− mouse model for sclerosing cholangitis alpha‐smooth muscle actin‐positive (α‐SMA+) myofibroblasts are thought to play a pivotal role in hepatic fibrogenesis. The aim of this study was 2‐fold: (1) to demonstrate that the origin of an important fibrogenetic cell population is the BM; and (2) to investigate whether transplantation of BM (BM‐Tx) affects liver function, staging, and grading. Surrogate markers for fibrogenesis and regulation of hepatic stellate cells (HSC) as well as progenitor‐cell‐derived fibrocytes in liver tissue were analyzed by quantitative real‐time polymerase chain reaction (PCR) and immunohistology. After lethal irradiation of recipient mice, BM‐Tx was carried out by way of tail vein injection of BM cells from marker protein donors (green fluorescent protein, GFP+) or Abcb4−/− mice as control (syngeneic Tx). Parameters of liver function were assessed serologically and histologically. Activated HSC of α‐SMA+/CRP2+ phenotype were expressed in ≈50% of proliferating bile ducts, whereas fibrotic liver parenchyma showed no expression thereof. Epithelial mesenchymal transfer (EMT) was visualized in the areas of proliferating bile ducts. The hematopoietic origin of CD34+ fibrocytes was demonstrated immunohistologically in livers of BM chimeric mice. These CD34+ cells infiltrated hepatic lobules from portal fields and developed a desmin+ phenotype expressing collagen type I in fibrotic parenchyma as well as in vitro after isolation by magnetic cell separation. Transplantation of GFP+/Abcb4+ BM improved liver function and staging compared with sham transplantation, but no significant differences were noticed among allogeneic and syngeneic Tx. Conclusion: The present study is the first to identify that both BM‐derived fibrocytes and HSC are involved in biliary fibrogenesis in Abcb4−/− mice. Our data suggest that changes in immunity subsequent to BM‐Tx may alter hepatic fibrosis. (HEPATOLOGY 2009.)

Neuropeptide Y Is Expressed by Rat Mononuclear Blood Leukocytes and Strongly Down-Regulated during Inflammation

Neuropeptide Y (NPY), a classical sympathetic comediator, regulates immunological functions including T cell activation and migration of blood leukocytes. A NPY-mediated neuroimmune cross-talk is well conceivable in sympathetically innervated tissues. In denervated, e.g., transplanted organs, however, leukocyte function is not fundamentally disturbed. Thus, we hypothesized that NPY is expressed by blood leukocytes themselves and regulated during inflammation. NPY mRNA and peptide expression were analyzed in mononuclear leukocytes isolated from the blood vessels of healthy rat kidneys, as well as from the blood vessels of isogeneic and allogeneic renal grafts transplanted in the Dark Agouti to Lewis or in the Fischer 344 to Lewis rat strain combination. Depending on the donor strain, acute allograft rejection is either fatal or reversible but both experimental models are characterized by massive accumulation of intravascular leukocytes. Leukocytes, predominantly monocytes, isolated from the blood vessels of untreated kidneys and isografts expressed high amounts of NPY mRNA and peptide, similar to expression levels in sympathetic ganglia. During acute allograft rejection, leukocytic NPY expression drastically dropped to ∼1% of control levels in both rat strain combinations. In conclusion, NPY is an abundantly produced and tightly regulated cytokine of mononuclear blood leukocytes.

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.

Acetylcholine and chronic vasculopathy in rat renal allografts.

Background. Chronic allograft vasculopathy (CAV) is an important aspect of chronic allograft injury, which limits the long-term success of renal transplantation. The pathogenesis of CAV is ill defined, and no effective therapies exist. Acute rejection episodes are a major risk factor for CAV. Recently, we demonstrated that leukocytes, which strongly accumulate in allograft blood vessels during fatal acute rejection, produce acetylcholine (ACh), which has the potential to provoke CAV. Herein, we test the hypothesis that ACh is also produced by leukocytes during the development of CAV. Methods. Kidneys were transplanted in the Fischer 344 to Lewis rat strain combination, an established experimental model for CAV. Isografts were performed in Lewis rats. The capacity of intravascular graft leukocytes to synthesize ACh was investigated during reversible acute rejection on day 9 posttransplantation and during the process of vascular remodeling on day 42. Furthermore, allograft recipients were treated with rivastigmine, which blocks enzymatic degradation of ACh. Results. The protein expression of the high-affinity choline transporter-1 and choline acetyltransferase was increased in leukocytes from allografts on day 9 and 42 posttransplantation. In addition, leukocytes accumulating in the lumina of allograft blood vessels were by far more numerous compared with isografts. In line with our hypothesis, ACh itself was detected by high-pressure liquid chromatography in graft leukocytes but not in leukocytes from untreated kidneys. Treatment with rivastigmine drastically exacerbated CAV compared with placebo. Conclusion. We suggest that endogenous ACh contributes to the pathogenesis of CAV and may be a promising target for novel therapies preventing CAV.

The Risk of Polyomavirus-Associated Graft Nephropathy Is Increased by a Combined Suppression of CD8 and CD4 Cell-Dependent Immune Effects

Polyomavirus-associated graft nephropathy (PAN) has emerged as a significant risk factor for kidney graft loss. We analyzed intracellular cytokine responses for possible protective versus permissive immunologic effects on BK-virus replication. One hundred five renal transplant patients included in a prospective single-center study were randomized to receive cyclosporine mycophenolate mofetil (MMF) (CM: n = 31), tacrolimus (Tac)/MMF (TM: n = 32) or Tac/MMF with conversion to everolimus (TErl; n = 32). Ten patients were not randomized (NR) due to contraindications to MMF. The immunosuppressive therapy was monitored pre- and posttransplantation at 4, 12, and 24 months using triple fluorescence flow cytometry for intracellular interleukin (Il)-2 Il-4 and interferon (IFN)-γ production in phorbol myristate acetate- and lipopolysaccharide- stimulated lymphocyte cultures. BK viremia screening was performed by reverse-transcriptase polymerase chain reaction testing on days 0, 14, 30, 60, 90, 120, 180, 270, 360, and 720. Seven of 105 (6.7%) patients developed biopsy-proven PAN (CM: n = 1, TM: n = 3, TErl: n = 2, NR: n = 1), among whom 4 lost their grafts (TM: n = 1, TErl: n = 2, NR: n = 1). Twenty-one of 105 (20.0%) patients had documented BK viremia. BK viremia which preceded PAN in all cases, was significantly associated with TM immunosuppression: 4/31 (12.9%) CM: 11/32 (34.4%) TM; 5/32 (15.6%) TErl, and 1/10 (10.0%) NR patients (P = .034). BK-viremic patients showed significantly diminished CD8(+) T-cell Il-2 production at 120 days (P = .011) and 1 year posttransplantation (P = .014) compared with non-BK-viremic patients. Patients with PAN displayed significantly lower CD4(+) T-cell Il-4 responses at 1 and 2 years after transplantation (1 year: P = .007; 2 years: P = .001) with diminished IFN-γ responses at 1 year after transplantation (P = .011). Our analysis showed the incidence of BK viremia to be increased among patients with defective cytotoxic CD8(+) T-cell -dependent immune reactivity. Recipients who progressed from BK viremia to overt PAN showed an additional immunologic defect in CD4(+) T-cell function. Patients on a Tac- plus MMF-based immunosuppression were at higher risk to develop BK viremia.

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.

Leukocyte accumulation in graft blood vessels during self-limiting acute rejection of rat kidneys.

During self-limiting acute rejection preceding chronic vasculopathy, large amounts of leukocytes, predominantly monocytes, interact with the endothelium of renal allografts. We aim to characterize them and to identify targets for functional and interventional studies. Leukocytes were harvested by vascular perfusion from Fischer 344 to Lewis renal allografts or Lewis isografts, followed by flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Leukocyte accumulation peaked in allografts on day 9. The percentage of monocytes expressing MHC class II and CD161 was increased whereas CD4, CD11a, CD43, and CD71 expression remained unchanged. IFN-γ, IL-1β, IL-2, IL-10, TNF-α, and iNOS mRNA increased in allograft leukocytes but IL-4, IL-6, IL-12, TGF-β, and tissue factor did not. During acute rejection, 1783 genes were differentially expressed. In conclusion, graft blood leukocytes display a unique state of partial activation during self-limiting rejection. Numerous differentially expressed genes deserve further investigation as potential factors in deciding the fate of the allograft.

Reduced expression of arrestin beta 2 by graft monocytes during acute rejection of rat kidneys

During acute rejection, numerous pro-inflammatory and cytotoxic monocytes accumulate in the vasculature of experimental renal allografts. Arrestins (ARRBs) are cellular regulators of inflammation, but nothing is known about their expression during rejection. Intravascular mononuclear graft leukocytes were isolated 4 days after kidney transplantation. ARRB1 and ARRB2 mRNA expression was reduced in blood leukocytes from allografts undergoing acute rejection, whereas on the protein level only ARRB2 was changed. Flow cytometry and confocal microscopy revealed ARRB1 and ARRB2 expression by monocytes and T cells, with a selective decrease in ARRB2 expression in monocytes during acute rejection. I-κB directly interacted with ARRB2 and the levels of both proteins strongly correlated. Concomitantly, the mRNA expression of NF-κB targeted genes increased. Our results suggest that activation of blood monocytes in renal isografts is dampened by high ARRB2 levels. During acute rejection, ARRB2 levels are reduced and classical monocyte activation is enabled via NF-κB activation.

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.