[Show abstract][Hide abstract] ABSTRACT: Dysregulation of dendritic cell (DC) mediated immune responses towards auto-antigens, is considered an important feature in the maintenance of experimentally induced heart failure (HF). In order to evaluate the role of blood DCs in cardiomyopathies of different origins, we examined myeloid (mDC) and plasmacytoid (pDC) subset levels and maturation characteristics, according to HF severity and etiology in humans.
Absolute numbers of mDCs and pDCs in 12 New York Heart Association (NYHA) class-II, 28 NYHA class III-IV HF patients and 18 healthy controls, were studied by 4-colour whole blood flow cytometry.
End-stage (NYHA III-IV) HF patients had comparable circulating DC subset levels to NYHA-II patients and controls. However, within the NYHA III-IV group total DC levels in patients with non-ischemic dilated cardiomyopathy (DCM) were higher (P<0.001) than in patients with coronary artery disease (CAD), hypertrophic cardiomyopathy (HCM) or other HF etiology. This was due to a significant increase of primarily mDCs (P<0.0001) and to a lesser extent of pDCs (P<0.05) in idiopathic DCM patients, independent of systolic or diastolic cardiac dysfunction. Maturation marker CD83 and lymphoid homing chemokine receptor CCR7 surface expression was enhanced only on mDCs, but not pDCs from DCM patients (P<0.05), compared to patients with CAD, HCM or other underlying cardiac pathophysiology.
Total blood DC levels in end-stage HF are elevated in patients with DCM. Whole blood DC characterisation may lead to new insights into the pathophysiology of idiopathic DCM in humans.
International journal of cardiology 02/2008; 131(2):246-56. DOI:10.1016/j.ijcard.2007.10.031 · 6.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been postulated that the plasmacytoid/myeloid dendritic cell ratio (pDC/mDC) reflects immune reactivity, and can therefore be used to monitor transplant recipients. We investigated the influence of Ficoll-Paque separation and PBMC cryopreservation on the pDC/mDC ratio and the expression of maturation markers, e.g. chemokine receptors (CKRs) CCR7, CXCR4, and CCR5, in comparison to fresh blood cells. Fractions of pDCs and mDCs, and CKR expression were measured by flow cytometry in fresh blood, in Ficoll-isolated PBMCs and in cryopreserved PBMCs from healthy individuals and kidney transplant recipients. Ficoll-isolation of PBMCs resulted in higher pDC/mDC ratios in both groups compared to fresh blood cells resulting from a relatively large increase in pDCs compared to mDCs. The pDC/mDC ratio increased further after cryopreservation of PBMCs from kidney transplant recipients. Ficoll-isolation and cryopreservation of PBMCs affected the proportion of mDCs and pDCs positive for CKRs, and their expression levels resulting in a more mature phenotype. In conclusion, the pDC/mDC ratio and pDC or mDC maturation status based on CKR expression, is dependent on manipulation of PBMCs. Therefore, fresh blood is preferable for monitoring purposes in transplant patients, as only these cells reflect the in vivo immune-status of patients accurately.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) are antigen-presenting cells that are pivotal for the initiation of the primary immune response. Patients with chronic kidney disease (CKD) with or without chronic intermittent haemodialysis (CIHD) show an impaired immune response. Dysfunction of DCs may underlie this phenomenon.
In this study, several different functions of monocyte-derived DCs (moDC) of patients with CKD class IV-V (glomerular filtration rate <30 ml/min) and patients on CIHD were studied in vitro and compared with age- and sex-matched healthy volunteers.
We demonstrate that, independent of the maturation stimulus used, mature moDC from both groups of patients did not acquire the same level of terminal differentiation as moDC from controls, as shown by analysis of cell surface markers and the relative high macropinocytosis activity of moDC. The stimulation of allogeneic T-cells by immature moDC and mature moDC did not differ between patients and controls. However, in the presence of immature moDC or antigen-loaded maturated moDC from patients, less proliferation of autologous T-cells was observed in response to recall antigens. There was no difference between moDC from controls and patients in their ability to activate naive T-cells and to differentiate them into Th1 and Th2 cells.
These results show that the terminal differentiation of moDC in patients with severe CKD is impaired. This impairment is not restricted to one maturation stimulus and is independent of treatment with haemodialysis.
[Show abstract][Hide abstract] ABSTRACT: FTY720 alters lymphocyte recirculation and homing by interfering with S1P receptors on lymphocytes, possibly in combination with chemokine receptors, and induces a decrease in PBL counts. In fresh, whole blood samples of 14 kidney transplant patients, we analyzed by flow cytometry the effect of FTY on the number of NK cells, monocytes, naïve (CCR7+) T cells, memory (CCR5+) T cells and B cells. Patients treated with 0.5, 2.5 or 5mg FTY/day showed a strong decrease in T and B cell numbers. NK cells and monocytes were not affected. FTY reduced primarily naïve T cells. From the memory T cells (CCR5+), predominantly CD8 cells, 40-60% remained in the circulation. The majority of the CCR7+ cells disappeared from the circulation within 3-6h, while a further reduction was achieved later. The more slowly decrease in naïve CCR7+ T cell numbers was also observed in the group treated with 0.25mg FTY/day. Elispot assays revealed no IL-4 producing cells and a low frequency of IFN-gamma producing cells. We suggest that both CCR7 dependent and independent mechanisms are involved in the depletion of T cells from peripheral blood.
[Show abstract][Hide abstract] ABSTRACT: Chemokines and their receptors have been implicated in the pathogenesis of different forms of heart failure (HF). We examined CC- and CXC-chemokine receptor expression in fresh peripheral blood leukocyte populations from 24 end-stage HF patients consisting of coronary artery disease (CAD; n = 6) and hypertrophic cardiomyopathy (HCM; n = 7) or idiopathic dilated cardiomyopathy (IDCM; n = 8) or valvular disease (VD; n = 3) and compared the data with 18 healthy controls. Levels of CCR1, 2, 3, 4, 5, and 7, and CXCR1, 2, 3, and 4 were measured by flow cytometry, and the expression profile was assessed as molecules of equivalent soluble fluorochrome units as well as frequency (percentage) of CD3+, CD4+, and CD8+ T cells and monocytes or granulocytes. Frequency of CD3+ CXCR4+, CD3+ CXCR1+, and CD3+ CXCR3+ cells was significantly increased in HF patients, whereas only CCR7 and CXCR4 expression levels were elevated on CD3+ cells. Both CD4+ CXCR4+ and CD8+ CXCR4+ cell frequencies were significantly increased irrespective of cardiac disease etiology. Elevated CCR7 expression was less pronounced on CD4+ than CD8+ cells in patients with CAD and IDCM. Expression of CXCR4 on CD8+ cells was upregulated substantially, regardless of the cause of disease. CD8+ CXCR1+ and CD8+ CXCR3+ but not CD4+ CXCR1+ or CD4+ CXCR3+ cells were increased in the HF patients with IDCM and CAD, respectively. Expression of CXCR1 or CXCR3 on both CD4+ and CD8+ cells did not differ in all the groups. For monocytes, frequency of CD14+ CCR1+ and CD14+ CCR2+ cells was significantly decreased in CAD patients, whereas, increase in CD14+ CXCR4+ cell frequency was accompanied with elevated CXCR4 expression. On granulocytes, CXCR1 and CXCR2 receptors were downregulated in all patients, compared with controls. Our results suggest that the altered expression profile of CC- and CXC-chemokine receptors on circulating leukocyte populations involves enhanced activation of the immune system, perhaps as part of the pathogenic mechanisms in HF. Modulation of the chemokine network could offer interesting novel therapeutic modalities for end-stage HF.
[Show abstract][Hide abstract] ABSTRACT: In a prospective study, calcineurin inhibitors (CNI) were withdrawn in patients two years after kidney transplantation. We questioned whether stopping CNI had an effect on the donor-specific reactivity, as CNI might hinder immune responses leading to graft acceptance.
We measured the donor-specific cytotoxic T lymphocyte (CTL) precursor frequency (CTLpf) in 54 patients before and after withdrawal of CNI. In addition, the T-cell reactivity of PBMC to donor and third-party antigens was tested in MLR, and in IFNgamma-Elispot. Reactivity to tetanus toxoid (TET) was studied as well.
Donor-specific CTLpf significantly decreased after CNI withdrawal (P=0.0001). In contrast, no difference was observed in third-party reactive CTLpf, donor and third-party reactive MLR and IFNgamma-Elispot. Proliferative responses and the number of IFNgamma-producing cells to TET also decreased after CNI withdrawal. The decrease in CTLpf correlated with the time between the two blood samples (before and after stopping CNI, P=0.05). This decrease was caused by stopping CNI, because there was no correlation between CTLpf and the duration of the CNI treatment after transplantation. Moreover, the percentage of regulatory T cells in the peripheral blood increased after CNI withdrawal.
We report here that after withdrawal of CNI the donor-specific CTLpf decreases. We hypothesize that CNI suppress regulatory mechanisms that have the potential to down-regulate donor-specific CTL responses and reactivity to TET.
[Show abstract][Hide abstract] ABSTRACT: Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)-gamma producing cells determined by enzyme-linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN-gamma producing cells reactive to this pathway increased significantly (P = 0.04) during AR and the number decreased (P = 0.005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN-gamma producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.
[Show abstract][Hide abstract] ABSTRACT: The effects of immunosuppressive agents on T cell function have been well characterized but virtually nothing is known about the effects of renal transplantation on human dendritic cells (DCs). With the use of flow cytometry, we studied the kinetics of myeloid and plasmacytoid DCs in peripheral blood of 24 kidney allograft recipients before and after transplantation, and in 23 donors before and after kidney donation. All patients were treated with tacrolimus, mycophenolate mofetil and prednisone. Surgery resulted in a strong decline in the number of myeloid and plasmacytoid DCs, both in kidney donors and in their recipients. However, in donors this effect was transient, as the numbers of both DC subsets had normalized completely by the third postoperative month. In contrast, the recovery of myeloid DC counts in kidney transplant recipients was only incomplete at the end of the 3-month follow-up, despite tapering of immunosuppression. The seven patients who required additional immunosuppressive treatment because of acute rejection experienced an even more marked decrease in DC counts in the early postoperative period compared with patients who remained rejection-free. Surgical procedures markedly affect the numbers of circulating myeloid and plasmacytoid DCs. Immunosuppressive drugs have important additional in vivo effects on this cell type and impair the reconstitution of the myeloid DC subset in peripheral blood after renal transplantation.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cell (DC) mediated allo-antigen presentation to host antigen specific T-lymphocytes initiates acute allograft rejection. We investigated peripheral blood DC (PBDC) incidence and DC subset reconstitution in relation to histological diagnosis of acute cellular rejection (AR) and administration of rejection therapy after clinical heart transplantation (post-HTx).
Venous blood from 20 HTx recipients under standard immunosuppression was collected during serial endomyocardial biopsy (EMB) prior to administration of rejection therapy in a 9-month follow-up post-HTx. Echocardiographic assessment of allograft function during EMB was performed to distinguish clinical necessity for rejection therapy within histologically rejecting patients (R). Myeloid (mDC) and plasmacytoid (pDC) subsets identified by flow-cytometry were analysed for different ISHLT rejection grades. Circulating PBDC incidence and mDC/pDC ratio were compared sequentially between non-rejecting (NR) recipients and R patients treated (3A(+)) or not-treated (3A(-)) with rejection therapy during follow-up.
Eleven samples from biopsy-proven AR episodes (AR(+): ISHLT>or=3) were compared to 89 samples from non-rejection episodes (AR(-): ISHLT grade 0, n=52; grade 1, n=29; grade 2, n=8). We observed an inverse correlation of mDCs (P<0.05) but not pDCs with increasing rejection grade. PBDC incidence and mDC/pDC ratio were low in blood samples obtained during AR (P<0.05 and P<0.01, respectively). Both PBDCs and mDC/pDC ratio decreased during each AR episode (P<0.05). Comparison of 3A(+) and 3A(-) rejectors with NR patients after 12 weeks post-HTx revealed lower PBDC incidence (P<0.01) and mDC/pDC ratio (P<0.05) for R patients, independent of rejection therapy.
Defective DC subset reconstitution by dendritic cell profiling identifies patients at risk for AR after 3 months post-HTx. This finding may contribute to further optimization of immunosuppressive treatment strategies after clinical heart transplantation.
European Journal of Cardio-Thoracic Surgery 05/2005; 27(5):783-9. DOI:10.1016/j.ejcts.2004.12.032 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allo-Ag presentation to Ag-specific T-lymphocytes by donor or recipient dendritic cells (DCs) induces acute rejection (AR) after solid organ transplantation. It is postulated that myeloid (mDC) and plasmacytoid (pDC) subsets circulate differentially between bone marrow, heart and lymphoid tissues after cardiac transplantation (HTx). We investigated peripheral blood DC subset distribution, maturation and lymphoid homing properties in relation to endomyocardial biopsy (EMB) rejection grade after clinical HTx. Twenty-one HTx recipients under standard immunosuppression were studied in a 9-month follow-up. mDC and pDC numbers were analyzed by flow cytometry in fresh venous whole blood samples collected during the EMB procedures and before histological diagnosis of AR. Subsets were further characterized for maturation marker CD83 and lymphoid homing chemokine receptor CCR7. Although numbers of both DC subsets remained low for the whole post-HTx period, we observed a negative association of mDCs with rejection grade. Repeated measurements analysis revealed that only mDCs decreased during AR episodes. Rejectors had lower mDC numbers after a 3-month follow-up compared to nonrejectors. Furthermore, patients during AR exhibited low proportions of mDCs positive for CD83 or CCR7. These findings suggest peripheral blood mDC depletion in association with selective lymphoid homing of this subset during AR after clinical HTx.
American Journal of Transplantation 05/2005; 5(4 Pt 1):810-20. DOI:10.1111/j.1600-6143.2005.00777.x · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dysfunctional antigen presentation may underlie the impaired antibody response to hepatitis B vaccination in hemodialysis patients. Dendritic cells are considered to be the most important antigen presenting cells, but their presence and function in hemodialysis patients is unclear. Granulocyte-monocyte-colony stimulating factor (GM-CSF) has been given successfully to hemodialysis patients to increase the proportion of responders to hepatitis B vaccination. Although GM-CSF acts on both monocytes and dendritic cells, the mechanisms underlying its adjuvant quality are largely unknown.
In this study we analyzed monocytes and dendritic cells in the peripheral blood of hemodialysis patient that had responded to a standard hepatitis B vaccination procedure (responders), patients who had not responded (nonresponders), and healthy controls. The nonresponders were given two additional booster vaccines, both preceded by administration of GM-CSF the day before.
After two booster vaccinations with GM-CSF, six out of seven patients developed a protective antibody response to hepatitis B. The memory T-cell response to tetanus toxoid was significantly lower in nonresponders compared to controls. The monocytes of dialysis patients and healthy controls showed a similar expression of relevant cell surface molecules. However, the numbers of circulating dendritic cells were on average 50% reduced compared to healthy controls, with a further reduction after GM-CSF administration. This was accompanied by a decrease of T-cell proliferation in antigen presentation assays. Monocytes showed increased major histocompatibility complex (MHC) class II, CD54, and CD40 expression, while their antigen-presenting capacity remained unchanged.
GM-CSF is an effective adjuvant for hepatitis B vaccination in primary nonresponding hemodialysis patients, but paradoxically decreases the antigen presenting capacity of peripheral blood mononuclear cells and the number of circulating dendritic cells.
Kidney International 09/2004; 66(2):614-21. DOI:10.1111/j.1523-1755.2004.00781.x · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) are antigen presenting cells that play a central role in inflammation, allograft rejection and immune tolerance. Myeloid (mDC) and plasmacytoid (pDC) subsets regulate immune reactions by polarising naive T-helper cells into a Th1 or Th2 response, respectively. In this study we examined total peripheral blood DCs, mDC and pDC subsets in chronic heart failure (CHF) and clinical heart transplantation (HTx).
We compared 16 heart transplant patients before and after HTx to 14 healthy controls. Whole blood was collected pre-HTx and 1-week post-HTx from patients and at corresponding time-points from controls. All patients received induction and maintenance immunosuppression post-HTx. mDCs and pDCs were measured by flow cytometry and were further characterised for maturation and homing potential to the secondary lymphoid organs with CD83 and CCR7, respectively. Data were expressed as absolute numbers/microl whole blood, percentage (%) mDC or pDC of total blood DCs and % positive DCs for CD83 and CCR7.
CHF patients had more peripheral blood DCs compared to controls (P<0.01) while only the mDC fraction was increased compared to controls (P=0.01). Percentage CD83(+) and CCR7(+) mDCs was also higher than control levels (P<0.05). One week post-HTx, total DCs, mDCs and pDCs decreased below controls (P<0.001). At the same time % mDCs in peripheral blood increased markedly compared to CHF and control levels (P<0.001). The %CD83(+) mDC, %CD83(+) pDC and %CCR7(+) mDC also returned to control levels and only %CCR7(+) pDC decreased below control levels (P=0.005).
Total peripheral blood DCs are elevated during CHF due to an increase in the mature fraction of the mDC subset suggesting a possible Th1 response in end-stage heart failure. The decrease in total DCs and mature mDCs and pDCs seen post-HTx, probably reflects immunological quiescence through adequate immunosuppression. Peripheral blood DC monitoring may provide a new insight into mechanisms of heart failure and allograft rejection by safe weaning from immunosuppression after clinical HTx.
European Journal of Cardio-Thoracic Surgery 05/2004; 25(4):619-26. DOI:10.1016/j.ejcts.2004.01.032 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In patients on chronic hemodialysis leukocyte activation has been related to the impaired function of the immune system. In this study we investigated if the vitamin E-coated dialyzer membrane could reduce monocyte activation thereby improving cellular immunity.
This hypothesis was tested in a prospective crossover trial in which 14 stable hemodialysis patients were switched from the baseline hemophane dialyzer to a vitamin E-coated and thereafter a polysulphone dialyzer membrane or vice versa.
Monocyte MHC class I, CD54 and ICAM-1 expression was significantly downregulated when a vitamin E-coated or polysulphone dialyzer was used. The use of a vitamin E membrane specifically decreased monocyte CD40 and CD86 expression. Lectin induced T cell proliferation increased with the use of the vitamin E-coated membrane as compared to polysulphone and hemophane dialyzers.
Vitamin E-coated dialyzers induced a less-activated phenotype of monocytes and may improve cellular immunity.