L. A. Van Es

AUTOANTIBODIES AGAINST NEUTROPHILS AND MONOCYTES: TOOL FOR DIAGNOSIS AND MARKER OF DISEASE ACTIVITY IN WEGENER'S GRANULOMATOSIS

Immunoglobulin G (IgG) autoantibodies against extranuclear components of polymorphonuclear granulocytes were detected in 25 of 27 serum samples from patients with active Wegeners granulomatosis and in only 4 of 32 samples from patients without signs of disease activity. In a prospective study of 19 patients these antibodies proved to be better markers of disease activity than several other laboratory measurements used previously. The autoantibodies were disease specific and the titres were related to the results of an in-vitro granulocyte phagocytosis test, in which 7S IgG antibodies were internalised after specific binding to the cell, resulting in gradual formation of ring-like cytoplasmic structures. This autoantibody may have a pathogenetic role in Wegeners granulomatosis. The detection of this antibody is valuable for diagnosis and estimation of disease activity.

Beneficial effects of conversion from cyclosporin to azathioprine after kidney transplantation

Immunosuppression with cyclosporin after renal transplantation is associated with better graft survival than is azathioprine treatment. However, nephrotoxicity and other side-effects have led to regimens that change treatment to azathioprine shortly after transplantation. Conversion has beneficial effects in the short term on renal function and hypertension. We report long-term follow-up (minimum 5 years) of 128 patients who had received a first or second cadaveric kidney graft and were treated with cyclosporin and prednisone; they were randomly assigned 3 months after transplantation to groups continuing to receive cyclosporin (n = 68) or changing to azathioprine (n = 60). 8 years after transplantation, patient survival was 75.3% in the cyclosporin group and 85.9% in the azathioprine group (p = 0.14) and graft survival was 64.0% and 76.6%, respectively (p = 0.38). The frequency of cardiovascular death with a functioning graft was 8% higher in the cyclosporin group (95% CI -1 to 18). The relative risk of graft loss after conversion to azathioprine compared with graft loss after conversion to azathioprine compared with cyclosporin maintenance was 0.71 (0.37-1.38) and the relative risk of patient death was 0.57 (0.23-1.41). The cyclosporin group had poorer mean creatinine clearance (17.8 mL/min [8.1-27.5] lower than azathioprine group) and a higher proportion needed hypertensive drugs (20% [4-36] more). Gout was found in 9 cyclosporin-treated patients and 1 azathioprine-treated patient (difference 12% [3 to 20]). Elective conversion from cyclosporin to azathioprine 3 months after transplantation is safe and cost-effective.

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.

European therapeutic trials in ANCA-associated systemic vasculitis: disease scoring, consensus regimens and proposed clinical trials EUROPEAN COMMUNITY STUDY GROUP ON CLINICAL TRIALS IN SYSTEMIC VASCULITIS ECSYSVASTRIAL (BMHl-Cr93-1078)

In previous phases of this project, proteinase 3 (PR3) and myeloperoxidase (MPO), the main antigenic target molecules of antineutrophil cytopiasmic antibodies, were isolated and applied in standardized ELISAs. In this study, standardized ELISAs with three PR3 preparations (from Copenhagen (CO), Raisdorf (RS) and Leiden (LF)) and one MPO preparation (from Copenhagen), were evaluated in a large retro-and prospective clitiical study. New patients (n=174) with primary systemic vasculitis (Wegeners granulomatosis, microscopic polyangiitis and idiopathic rapidly progressive glomerulonephritis, classical PAN and Churg-Strauss Syndrome) were included. Retrospectively, another 190 patients were evaluated. Furthermore control sera were obtained from patients with other forms of vasculitis, glomerulonephritis or granulomatous diseases (disease controls, n = 184) and healthy donors (healthy controls, n = 728). All patients were categorized by a system based on clinical and histoiogical data. Patients were followed up for at least 1 year after diagnosis in order to evaluate a possible correlation between ANCA levels and disease activity. The sensitivity of the anti-PR3 assays for histologically proven WG was between 59% and 69% in new patients, with a sensitivity of 22% for the anti-MPO assay. Similar figures were found for patients with clinically suspected WG. This was comparable with the results of the IIF test. In MPA and IRPGN a larger percentage of patients had antiMPO antibodies than in WG. Only a few patients with PAN and CSS were investigated, and most of these were negative in the ELISAs. The specificity ofthe assays for disease controls was 89-91% for the anti-PR3 assays and 95% for the anti-MPO assay. In the healthy controls the specificity was 98-99%. The specificity of the IIF test was 97% for a cANCA pattern and 81 % for a pANCA pattern in disease controls. The combination of cANCA with anti-PR3 and pANCA with anti-MPO both had a specificity of 99%. Further details will be presented during the meeting, in addition to the results of a follow-up study with correlation ofdisease activity and ANCA level. From this study we can conclude that ELISAs using purified PR3 or MPO are not more sensitive than the IIF test. However, the anti-MPO assay is more specific for systemic vascuitis as compared to disease controls with related diseases. Furthermore, the combination of the IIF test with antigen-specific ELISAs is very specific for the diagnosis Wegetiers granulomatosis, microscopic polyangiitis and idiopathic rapidly progressive gtomerulonephritis.

Regulation and production of IL‐8 by human proximal tubular epithelial cells in vitro

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL‐8. In the present study we have analysed the production of IL‐8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum‐free conditions, were found to produce IL‐8 to different degrees from not detectable levels up to 10.8±1.5 ng IL‐8 per 1×105 cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL‐8 of PTEC is 15.1 and 8.1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0.5 ng/ml rIL‐1α or 1000 U/ml recombinant tumour necrosis factor‐alpha (TNF‐α) to the culture media of PTEC induced an up‐regulation of IL‐8 production up to 6.3‐fold and 3.0‐fold, respectively. The up‐regulation by IL‐1α and TNF‐α was dose‐ and time‐dependent. In contrast, 500 U/ml recombinant interferon‐gamma (rIFN‐γ) down‐regulated the production of IL‐8 3.4‐fold. Northern blot analysis showed that IL‐1α and TNF‐α increased the expression of IL‐8 mRNA, whereas IFN‐γ reduced IL‐8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL‐8 in the kidney, and that IL‐8 produced in the proximal tubule can be induced by various proinflammatory cytokines.

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.

Humoral immune response to influenza vaccination in patients with primary immunoglobulin A nephropathy. An analysis of isotype distribution and size of the influenza-specific antibodies.

Primary IgA nephropathy (IgAN) is characterized by mesangial deposits of IgA1, increased serum IgA1 levels, and circulating immune complexes containing predominantly IgA1. It has previously been found that patients with IgAN have a higher than normal IgA response to vaccination, but the IgA subclasses have not been studied. To investigate whether the IgA hyperresponsiveness is limited to the subclass IgA1, which is involved in the pathogenesis of IgAN, we compared the immune responses of 18 patients with 22 healthy controls after intramuscular vaccination with inactivated influenza virus. Antibody titers were significantly higher (P less than 0.0001) for the IgA1 subclass in patients versus controls, but not for the other isotypes. A substantial portion of the IgA and IgA1 antiinfluenza immune response comprised polymers in both patients and controls. There was no preferential response of polymers in patients. Patients produced significantly more monomeric IgA1 antibodies than controls. These results show that patients with IgAN have a hyperresponsiveness limited to the subclass IgA1 and mainly expressed by an excess of monomers.

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.

Expression of HLA-DR on T lymphocytes following renal transplantation, and association with graft-rejection episodes and cytomegalovirus infection

The expression of HLA-DR by T lymphocyte subpopulations recognized by monoclonal antibodies and flow cytometry was monitored in 10 normal controls, 15 patients on hemodialysis, 25 recipients of a renal allograft with stable graft function, 16 transplant recipients suffering rejection episodes, and 4 transplant recipients with cytomegalovirus (CMV)4 infection. No significant differences in HLA-DR expression were observed in the OKT4-reactive subpopulation among these controls and patients. In the 16 patients who suffered rejection episodes a significant increase in HLA-DR expression by OKT8-reactive cells was observed. In the 10 patients in whom rejection episodes occurred later than 1 week after transplantation, the percentage of OKT8-reactive cells that expressed HLA-DR was dramatically increased 2-6 days before the onset of clinical symptoms of rejection (P less than 0.001 Students t test). When the rejection episode responded to treatment, expression of HLA-DR on the OKT8-reactive cells decreased--whereas in 3 patients who lost their grafts because of irreversible rejection, expression of HLA-DR remained elevated. In addition, the 4 patients with CMV infection had increased percentages of OKT8-reactive cells that expressed HLA-DR (P less than 0.001 Students t test). This increase in expression of HLA-DR coincided with the onset of clinical symptoms of the CMV infection, and the expression of HLA-DR returned to normal values after the titers of antibodies to CMV had increased significantly and symptoms of infection had disappeared.