F. J. Van Der Woude

Interleukin 2 mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.

Previous reports have suggested the production of complement components C4, C2, and factor B by renal tissue. However, the cells involved in production of complement have not been identified. In this study metabolic labeling experiments demonstrated that human proximal tubular epithelial cells (PTEC) synthesize a 180-kD precursor of C3 that is secreted after proteolytic cleavage into a disulphide linked two-chain molecule as found in plasma. C3 present in culture supernatants of PTEC was functionally active, however, during the culture period there was a partial inactivation of the C3 molecule as assessed by hemolytic titration. Recombinant IL-2 enhances the rate of C3 synthesis in a dose-dependent manner reaching maximal stimulation at doses of 200-400 U/ml IL-2. Northern blot analysis demonstrated a 5.2-kb C3 mRNA species present in PTEC that was increased within 24 h of IL-2 treatment. IL-2-induced enhancement of C3 production by PTEC could be neutralized with specific antibodies to IL-2. This study demonstrates that C3 synthesis in PTEC is upregulated by IL-2, the major cytokine produced by activated T cells.

Cell-mediated autoimmunity in patients with Wegener's granulomatosis (WG)

Despite the well described infiltration of cells of the cellular immune system in vasculitic lesions and the granuloma formation in patients with WG, the role of T cell‐mediated autoimmunity in WG is not clear. Reports of T cell proliferation in response to neutrophil azurophilic granule proteins are contradictory. In this study we have assessed the proliferation of T cells of WG patients to purified proteinase 3 (PR3) and to total azurophilic granule proteins in two different assays. In addition to the classical proliferation assay with isolated peripheral blood mononuclear cells, we have used a whole blood proliferation assay. In both assays we found proliferative responses to PR3 in patients with WG. The number of patients reacting to the azurophilic granule extract was higher than the patients reacting to the purified PR3, suggesting that other autoantigens may also be involved. We have identified epitopes of PR3 that may be potential targets of class I‐restricted T cell responses in the context of HLA‐A*0201, the most common MHC class I molecule. These epitopes were determined by the binding of synthetic PR3 peptides to HLA‐A*0201 on the antigen‐processing defective cell line, T2. In addition, T cell lines were established from tissue biopsies, obtained from WG patients, and assessed for cytolytic reactivity against T2 cells, preloaded with synthetic PR3 peptides. We conclude that T lymphocytes of WG patients have increased proliferative responses to purified PR3 and to a larger extent to non‐fractionated proteins of azurophilic granules of polymorphonuclear neutrophilic leucocytes (PMN).

The clinical significance of allospecific antibodies against endothelial cells detected with an antibody-dependent cellular cytotoxicity assay for vascular rejection and graft loss after renal transplantation.

Serum samples of 64 consecutive patients who underwent renal transplantation in our institution were examined for the presence of antibody-dependent cellular cytotoxicity (ADCC) activity against endothelial cells (EC). From each patient serum samples were obtained immediately before transplantation and 1 week, 1 month and 1 year thereafter. The results were evaluated in the context of tests to measure donor-specific humoral immunity against lymphocytes and monocytes, and related to parameters of presensitization, graft survival, and histology. Sera from 10 patients were positive for ADCC on a panel of HLA-typed endothelial cells. In 8 patients sera were already positive before transplantation and remained positive thereafter. In 4 patients a positive crossmatch with donor T and B cells and monocytes could be observed after transplantation. In only one patient were these crossmatches positive before transplantation. A significant correlation was found between ADCC positivity and vascular rejection (P=0.015); in addition graft survival was significantly better in the ADCC negative group vs. the positive group (P=0.0004). These data demonstrate the significance of allospecific anti EC antibodies for the occurrence of vascular rejection and graft loss after renal transplantation.

Regulation of glomerular epithelial cell production of fibronectin and transforming growth factor-beta by high glucose,not by angiotensin II

Accumulation of matrix proteins is a prominent feature of diabetic nephropathy. Glomerular visceral epithelial cells (GVECs) are important contributors to extracellular matrix (ECM) production in the glomerulus. Factors involved with increased accumulation of ECM proteins are high glucose, angiotensin II (ANG II), and transforming growth factor (TGF)-β. Therefore, we investigated the effects of high glucose and ANG II on fibronectin and TGF-β production by human GVECs in vitro. We found that ANG II had no effect on the production of fibronectin and TGF-β by GVECs. Using reverse transcriptase–polymerase chain reaction analysis, no ANG II receptor could be detected on these cells. However, high glucose induced a twofold increase in fibronectin (P < 0.01) and a three- to sixfold increase in TGF-β (P < 0.001) production. Similar results were obtained by analyzing the mRNA levels of fibronectin (increased 2.7-fold) and TGF-β (increased 3.5-fold). Addition of increasing concentrations of rTGF-β to control cells resulted in increased fibronectin production. Neutralizing antibodies against TGF-β significantly reversed the increase in fibronectin protein and mRNA caused by high glucose back to control levels. We conclude that high glucose concentrations stimulate the synthesis of fibronectin and that this effect is mediated by induction of TGF-β. These results suggest that in diabetic nephropathy, high glucose levels play a role in changing the matrix composition of the glomerular basement membrane through induction of TGF-β. Our results indicate that a contribution to this process by an effect of ANG II on GVECs seems unlikely.

Abnormalities of CD4+ T cell subpopulations in ANCA-associated vasculitis

In patients with ANCA‐associated vasculitis (AAV), CD25 expression is increased on circulating T cells. Although in animal experiments the role of CD4+ CD25+ T‐regulatory‐cells (Treg) in protection against autoimmunity is well established, the role of these cells in AAV is unknown. To investigate the hypothesis that an increased expression of CD25 on T cells is related to persistent T cell activation and not to disturbances in Treg cells in AAV (34 patients, six of them after renal transplantation), we investigated CD25 expression in different subpopulations of CD4+ cells and FOXP3 mRNA expression by reverse transcription‐polymerase chain reaction (RT‐PCR). In addition, T cell proliferation and cytokine secretion after stimulation with anti‐CD3 and anti‐CD28 and intracellular cytokine production after stimulation with phorbol myristate acetate (PMA)‐ionomycin was determined. Controls were non‐vasculitic renal transplant patients (n = 9) and healthy controls (HC) (n = 13). In AAV the total number of lymphocytes, CD4+ lymphocytes and the percentage of naive T cells are lower than in HC and RTX. An increased percentage of CD25+ cells was found in AAV and AAV/RTX, irrespective of disease activity, but not in HC or RTX. This was confined to the naive (CD4+ CD45RBhigh) population only. FOXP3 mRNA expression in CD4+ T cells did not differ between AAV patients and healthy controls. In vitro T cell proliferation was enhanced in AAV patients compared to HC (P < 0·01). PBMC of AAV patients produced significantly less interleukin (IL)‐10 and interferon (IFN)‐γ after anti‐CD3/CD28 stimulation. The percentage of IL‐10 and IL‐12, but not IFN‐γ, IL‐4 or tumour necrosis factor (TNF)‐α‐producing cells was significantly higher in patients compared to HC. These findings were confined to the memory population of CD4+ cells. We conclude that AAV patients are lymphopenic and have low numbers of CD4+ T cells, which seem to be in a persistent state of activation.

Donor-specific lysis of human kidney proximal tubular epithelial cells by renal allograft-infiltrating lymphocytes.

In the present study methods are described to obtain both graft infiltrating cells (GIC) of host origin and proximal tubular epithelial cells (PTEC) of donor origin simultaneously from biopsy material of renal allografts undergoing rejection. The identity of PTEC cultures was established using monoclonal antibodies. GIC were shown to exhibit T cell functional activity. These GIC were shown to lyse trypsinized PTEC as well as PTEC monolayers grown from the corresponding biopsy, and not PTEC isolated from biopsies obtained from other patients. Therefore the lytic activity appeared to be donor-specific. Major histocompatibility complex class I antigens were involved since donor PHA-blasts, a target population well known to express class I molecules, were lysed by GIC, and the anti-class I MoAb W6/32 blocked cytolytic activity of GIC against donor PHA-blasts and against donor PTEC. We thus established that donor-specific lysis of a defined population of kidney epithelial cells, namely PTEC, may occur. This model system, in which GIC and PTEC can be propagated from one biopsy specimen may be useful for further study of cell-cell interactions involved in allograft rejection.

Regulation of C3 and factor H synthesis of human glomerular mesangial cells by IL-1 and interferon-gamma.

Previous reports have shown production of complement components C4. C2 and factor B by renal tissue. We have shown recently that human proximal tubular epithelial cells (PTEC) synthesize C3 in vitro, and that IL‐2 enhances this production. In the present study we demonstrate that human mesangial cells (MC) in culture produce factor H and that supernatants of activated peripheral blood mononuclear cells (T cell growth factor (TCGF)) induce C3 production and enhance factor H synthesis in both a time‐ and dose‐dependent manner. To investigate whether certain defined cytokines from TCGE were responsible for the observed effect., we tested various cytokines for their effect on complement production by MC. It is shown that IL‐1 induces C3 synthesis whereas factor H production is up‐regulated by TFN‐γ, in both a dose‐ and time‐dependent manner. Antibody blocking experiments revealed that C3 synthesis induced by both TCGF and IL‐I could be blocked with antibodies specific for IL‐I, and also that TCGF and IFN‐γ enhanced factor H synthesis could both be blocked with antibodies specific for IFN‐γ. Cycloheximide was able to inhibit C3 and factor H production, suggesting de novo synthesis of the proteins. mRNA‐polymerase chain reaction (PCR) analysis revealed mRNA encoding for C3 after stimulation with TCG Fand IL‐I. Factor H genes are constitutively expressed in cultured mesangial cells and its expression is up‐regulated by TCGF and IFN‐γ. Northern blot analysis with specific probes for C3 and factor H revealed bands which support the results obtained by PCR analysis.

Long-term cardiovascular morbidity and mortality in autosomal dominant polycystic kidney disease patients after renal transplantation.

Patients with autosomal dominant polycystic kidney disease (ADPKD) have an increased incidence of hypertension and cardiovascular abnormalities. In this long-term follow-up study (5.88 years on average), we evaluated cardiovascular disease and patient and graft survival in 101 ADPKD patients and 692 nondiabetic control patients receiving cadaveric renal transplants between March 1967 and April 1991 at the Leiden University Hospital. Graft and patient survival was not different between patient groups, using the same immunosuppressive therapy. However, death with functioning graft, mainly due to cardiovascular disease, was significantly more frequent in the ADPKD patients than in controls using AZA (P < 0.01). Multivariate analysis of pretransplant data showed that ADPKD patients on AZA therapy demonstrated an elevated age-adjusted relative risk of 2.07 (95% confidence interval [95% CI]: 1.12-3.80) for cardiovascular events and 2.88 (95% CI: 1.41-5.90) for cardiovascular mortality alone. After adjustment for age, gender, and other cardiovascular risk factors, a relative risk of 2.39 (95% CI: 1.06-5.40) was found. This was 2.87 (95% CI: 1.04-7.93) when cardiovascular mortality was the dependent variable. With posttransplant data, the age-adjusted relative risk for cardiovascular morbidity and mortality in ADPKD patients using AZA was 2.16 (95% CI: 1.12-4.15) and 2.97 (95% CI: 1.40-6.27), with only cardiovascular mortality as the dependent variable. After adjustment for age, gender, and other cardiovascular risk factors, this was 1.59 (95% CI: 0.64-3.91) and 2.28 (95% CI: 0.79-6.53), respectively. With CsA treatment, an elevated risk for cardiovascular morbidity and mortality in ADPKD patients was present, but the corresponding 95% CI were wide and include unity, due to the shorter period of follow-up (CsA: 3.81 +/- 2.50 years vs. AZA: 7.28 +/- 6.74 years). Survival of ADPKD patients using AZA was less in those patients without pretransplant nephrectomy as compared with control patients, but the morbidity and mortality of pretransplant nephrectomies should be taken into account. We conclude that ADPKD patients show a similar graft and patient survival after renal transplantation as control patients, but they are especially at risk for cardiovascular disease after renal transplantation.

Cytomegalovirus directly enhances MHC class I and intercellular adhesion molecule-1 expression on cultured proximal tubular epithelial cells

Clinically, there is an association between CMV infections and the occurrence of rejection after renal transplantation. Adhesion molecules such as intercellular adhesion molecules (ICAM) and MHC Ags are thought to be important in the induction and amplification of the rejection process. Therefore, we studied ICAM-1 and MHC expression after CMV infection or stimulation with cytokines. Cultured proximal tubular epithelial cells (PTEC) were stimulated with cytokines or infected with CMV. MHC class I, class II, and ICAM-1 expression were determined by radioimmunoassay. IFN-7 induced class II and ICAM-1 expression. Small concentrations of IFN-α inhibited the IFN-7 induced class II expression. CMV-infected PTEC displayed increased levels of ICAM-1 and class I expression. This enhancement is a direct effect of the virus on the infected cells and not mediated by soluble factors. Although MHC class II expression is not directly enhanced by CMV, infected PTEC display a normal increase of class II expression after stimulation with IFN-γ.

Interstitial rejection, vascular rejection, and diffuse thrombosis of renal allografts : Predisposing factors, histology, immunohistochemistry, and relation to outcome

Histological and immunohistochemical analyses were made of biopsy specimens from 50 consecutive patients who experienced putative graft rejection. The mean age of the patients was 44.5 years (range, 17-69 years) and 26 were men. There were 67 evaluable allograft specimens, which were grouped according to the histological diagnosis: group 1, acute tubulointerstitial rejection (n = 42); group 2, acute vascular rejection (n = 18); and group 3, diffuse thrombosis (n = 7). Over a follow-up period of 21-57 months, the mean number of rejection episodes was 1.7, 2.8, and 3.3 in groups 1, 2, and 3, respectively. Allograft loss occurred in 7 out of 30, 10 out of 16, and 4 out of 4 patients in groups 1, 2, and 3, respectively. The following histological parameters differed significantly (P < 0.05) among the groups: interstitial edema, congestion of peritubular capillaries, glomerular thrombosis, and glomerular ischemia (group 3 > group 2 > group 1). Interstitial bleeding was seen more often in group 2 and 3 tissues than in group 1 specimens (P < 0.01). Immunohistochemical analyses showed that vascular rejection was associated with WT14 staining for monocytes and macrophages around the tubuli and with interstitial deposition of complement factor 3. With regard to serology, positive anti-endothelial cell antibody-dependent cellular cytotoxicity was associated with vascular rejection and thrombosis of the graft in all patients tested, and with graft loss in 75%. Pre-existent positive anti-IgG immunofluorescence on peritubular capillaries in pretransplant biopsy specimens incubated with patient serum was found in only 3 of the 50 patients, but was associated with graft loss in 2 of the 3. Cytomegalovirus infection was associated with a higher percentage of graft loss. There were significant intergroup differences in panel reactive antibodies before transplantation (P < 0.001), with higher titers in groups 2 and 3. The findings in relation to interstitial rejection are compatible with cellular rejection, while the data on vascular rejection support a humorally mediated pathogenesis.