Circulating cytokine profile in anti-neutrophilic cytoplasmatic autoantibody-associated vasculitis: prediction of outcome?

AIMS: The anti-neutrophilic cytoplasmatic autoantibody-associated vasculitides (AASV) are diseases of relapsing-remitting inflammation. Here we explore the cytokine profile in different phases of disease, looking for pathogenic clues of possible prognostic value. RESULTS: Interleukin (IL)-6, IL-8 and IL-10 were significantly elevated in plasma. Patients in the stable phase who subsequently developed adverse events had higher IL-8 values. Patients in the stable phase who relapsed within 3 months had lower IL-10 values and higher IL-6 levels. CONCLUSIONS: Patients with AASV have raised circulating cytokine levels compared with healthy controls, even during remission. Raised IL-8 seems associated with poor prognosis. Lower levels of IL-10 and higher levels of IL-6 herald a greater risk of relapse. Patients with systemic vasculitis in clinical remission have persistent disease activity, kept under control by inhibitory cytokines

Identification of a target antigen in human anti-tubular basement membrane nephritis

Identification of a target antigen in human anti-tubular basement membrane nephritis. Sera from two patients with primary anti-tubular–basement–membrane–mediated tubulointerstitial nephritis, one a renal allograft recipient and the other with spontaneous anti-tubular–basement–membrane disease, were analyzed for the specificity of their autoantibodies. Both sera had circulating antibodies that reacted by ELISA with extracts of tubular basement membrane from several species, but failed to react significantly with extracts of glomerular basement membrane. Reactive antigen was solubilized with 6 M guanidine-HCl, 6 M urea, with reduction and alkylation, and with sodium dodecylsulfate. Digestion of the basement membrane with collagenase released relatively small quantities of antigen from the membrane, and trypsin and pepsin destroyed its antigenicity. The antigenic activity was characterized with respect to its size distribution by gel filtration and by immuno-overlay analysis of protein blots. Collectively, the results indicate that the major reactivity of both sera is directed towards a Mr 58,000 component that is unique to the tubular basement membrane. Minor reactivities toward high molecular weight components common to both glomerular and tubular basement membranes were detected by immuno-overlay analysis. This study identifies an antigen that is involved in human anti-tubular–basement–membrane–mediated tubulointerstitial nephritis, and demonstrates an advantage of the use of denaturing extraction over proteolytic methods to prepare the antigen

Novel distribution of the secretory leucocyte proteinase inhibitor in kidney

THE secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue-specific inhibitor of, for example, elastase and cathepsin G, which also have antimicrobial capacity. SLPI has been localised to the respiratory, gastrointestinal and genital tracts, but so far not to the kidney. The presence of SLPI in renal tubuli cells was demonstrated using immunohistochemistry and, by means of in situ hybridisation on human renal biopsies, we were able to demonstrate SLPI production. In various inflammatory conditions in the kidneys, the protease-antiprotease balance is disturbed. For this reason, as well as the possible role in the defence against ascending urinary tract infections, it is interesting to establish a source of SLPI in renal tubuli cells. Key words: Glomerulonephritis, Kidney, Protease inhibitor, SLPI, Anti-leukoprotease Introduction Secretory leukocyte proteinase inhibitor (SLPI) is a 107 amino acid non-glycosylated single chain protein, stabilised by intrachain disulphide bonds. The estimated molecular weight of the acid-stable protein is 12 kDa. The C-terminal is responsible for the inhibitory activity against elastase, cathepsin G, chymotrypsin and trypsin, whereas the N-terminal has been reported to possess antimicrobial capacity. 1,2 SLPI is also known to inhibit HIV type 1 by blocking DNA synthesis. 3 The Kazal-type inhibitor forms complexes with the proteases and has a half-life of 10 min in the circulation. The complexes formed locally dissociate in plasma and the proteases are taken over by the plasma protease inhibitors (alpha-1-antitrypsin, alpha-2-macroglobulin and antichymotrypsin). 1 Furthermore, SLPI has been shown to inhibit elastin-bound elastase, which may be of importance for the preservation of elastic lung tissue. 7 The aim of the study presented here was to clarify whether there is a local renal production of SLPI. A possible local production would be of great interest, considering the multitude of inflammatory conditions, including different kinds of glomerulonephritis and nephritis, involving the kidneys. Anti-neutrophilic cytoplasmic antibodies (ANCA) are autoantibodies directed against constituents of polymorphonuclear neutrophils and monocytes, associated with systemic vasculitis and glomerulonephritis. One of the ANCA antigens, proteinase 3, has the capacity to attack and degrade components of the extracellular vessel wall matrix and the glomerular basement membrane. In blood, it is immediately bound, mainly by alpha-1-antitrypsin. Patients with ANCA-associated vasculitis have an over-representation of the PiZ gene of alpha-1-antitrypsin and thus a lack of the inhibitor. 8 SLPI has no direct inhibitory capacity against proteinase 3; it can, on the contrary, be degraded proteolytically by it. 1 Although SLPI has no direct inhibitory capacity against proteinase 3, the progressive binding of elastase to SLPI instead of alpha-1-antitrypsin has been shown to be paralleled by an increasing binding of Short Communication Mediators of Inflammation, 10, 347-350 (2001) proteinase 3 to alpha-1-antitrypsin. 10,11 A possible role in the defence against ascending urinary tract infections also draws attention to the point of issue. Subjects and methods Patient material Macroscopically and microscopically normal kidney preparations were selected from three different specimens obtained at the time of nephrectomy due to renal cancer. The Helsinki declaration regarding the use of human tissues was strictly observed. Materials and chemicals Normal rabbit serum was purchased from Dakopat AB (Copenhagen, Denmark). Swedish landrace goats were immunised with recombinant human SLPI, to produce anti-SLPI antiserum. Biotinylated rabbit anti-goat IgG and avidin-biotinylated horseradish peroxidase complexes were from Vector Laboratories (Burlingame, CA, USA). The AEC substrate-chromogen system and Faramount aqueous mounting medium were from Dako Corporation (Carpinteria, CA, USA). An oligonucleotide probe cocktail for SLPI was from R&D systems (Abingdon, UK). Blocking reagent and anti-digoxigenin (DIG)-AP conjugate (Fab fragments) were from Boehringer Mannheim (Mannheim, Germany). Visualising substrates (bromochloroindolylphosphate (BCIP) and nitroblue tetrazolium (NBT)) were from Bio-Rad Laboratories (Hercules, CA, USA). Immunohistochemistry The frozen renal tissue specimens were fixed in 4% buffered formaldehyde, dehydrated and embedded in paraffin wax for sectioning on sialinised slides. After rehydration, the sections were exposed to pepsin (4 mg/ml in 0.01 M HCl) for 20 min at 37°C. The sections were then washed between every step in Tris-buffered saline (TBS), 3 ´5 min. Endogenous peroxidase activity was removed by incubation of the specimens in 0.3% H 2 O 2 in methanol for 30 min at room temperature. Normal rabbit serum (3% in TBS) was applied to block non-specific staining. The next step, after draining, was to incubate the specimens with primary antibody (goat anti-SLPI, 1/1000, 30 min). For detection, we used biotinylated rabbit anti-goat IgG (5 mg/ml), followed by application of avidin-biotinylated horseradish peroxidase complexes for 30 min. Visualisation was accomplished by addition of AEC (0.75 mg/ml of 3-amino-9-ethylcarbazole in 2.5% N,N-dimethylformamide and 50 mM acetate buffer) that was left to incubate for 15 min. The slides were washed and counterstained before mounting. All the incubations were carried out at room temperature. Adsorbed anti-SLPI antiserum, obtained by affinity chromatography on a SLPI-conjugated Sepharose 4B-column, was used as a negative control and applied instead of the primary antibody. In situ hybridisation A cocktail of three 30-base long oligonucleotide probes (59-TCTTAGGAGGACAGACTC CAGCTTT-GAAGG-39, 59-ATTTCCCACACATGCCCATG CAA-CACTTCA-39, 59-AAC ATCTCTTCTTCCCTGGA-CACTGCCAGT-39), based on the antisense sequence of SLPI and labelled with DIG at the 59 end, was used for the in situ hybridisation. Diethylpyrocarbonate was added to the millipore water and all solutions to block RNAse activity. All solutions were autoclaved or sterilised by filtration. Renal tissue was taken immediately after surgery, put into liquid nitrogen and stored at -70°C. After sectioning on sialinised slides, the specimens were left to dry in air. After fixation (4% paraformaldehyde in 1 ´phosphate-buffered saline (PBS), 2 h, 4°C), the slides were incubated in a sucrose solution (30% sucrose in 1 ´PBS, overnight, 4°C). Washing in 1 Ṕ BS for 2 ´5 min was followed by treatment with 0.1 M glycine, 2 ´5 min. After further washing in 1 Ṕ BS for 2 ´5 min, the sections were placed in 0.3% Triton X-100 in 1 ´PBS for 15 min. Washing in 1 ´PBS, 2 ´5 min, was followed by incubation with proteinase K (1 mg/ml) in TE buffer for 30 min at 37°C. After post-fixation (4% paraformaldehyde in 1 ´PBS, 5 min, 4°C) and PBS washing, 2 ´5 min, the sections were acetylated in a 0.1 M triethanolamine (TEA) buffer (pH 8) with 0.25% acetic anhydride. TEA blocks endogenous activity of alkaline phosphatase, and acetanhydride reduces probe stickiness. Incubation with 40 ml pre-hybridisation buffer at 37°C for 2 h preceded the pre-hybridisation, which was performed in a moist chamber. To prevent evaporation, the specimens were covered with parafilm. The probe cocktail (10 ng in 40 ml of hybridisation buffer) was added and left to hybridise overnight at 37°C, which is 23°C below T m . This temperature was chosen because of short oligonucleotides. The slides were then washed in 2 ´SSC (2 5 min), 1 ´SSC (2 ´15 min) and 0.25 SSC (2 1 5 min) in a shaking water bath at 37°C. The slides were then immersed in buffer I (100 mM Tris-HCl, 150 mM NaCl; pH 7.5) for 2 ´10 min, followed by treatment with buffer II (buffer I + 0.5% blocking reagent), to block non-specific anti-DIG binding. The sections were then placed in a moist chamber and the anti-DIG-AP conjugate (1/500 in buffer I, 0.1% Triton X-100 and 1% normal sheep serum) was applied. Repetition of buffer I washing was carried out for 2 1 0 min. The slides were then placed in buffer III (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl 2 ; pH 9.5) for 10 min. Visualisation was achieved by incubation in 10 ml buffer III, 45 ml of NBT, 35 ml of BCIP and 1 mM Levamisole (240 mg/ml) for 2-4 h. The sections were then mounted using an aqueous mounting solution. Control sections were incubated with the matched sense-probes or with hybridisation buffer without probes. Results Immunohistochemical staining for SLPI was carried out on healthy kidney biopsies with positive staining results Discussion SLPI is considered to be a major protease inhibitor in the respiratory as well as in the gastrointestinal and genital tracts. A potential role in the urinary tract could therefore be anticipated. In this study, both immunohistochemistry and in situ hybridisation (ISH) showed clearly positive results, with no background staining and adequate controls. The positive staining of SLPI in distal tubuli in immunohistochemistry could be explained by renal filtration, but ISH also show a positive signal for SLPI mRNA in tubuli. These results imply local SLPI production in normal kidney, which is something that has not been demonstrated before. Our primary antibody has been used in many previous studies with clear-cut results, and must be considered highly specific. 12 For our ISH, we used an oligonucleotide cocktail synthesised for us by R&D Systems. These probes have worked well in previous studies, and the negative results obtained with the matched sense-strand probes confirm the specificity of the reaction for SLPI

Goodpasture disease. Characterization of a single conformational epitope as the target of pathogenic autoantibodies

Goodpasture disease is a prototype autoimmune disease characterized by the formation of autoantibodies against the heterotrimeric basement membrane collagen type IV, which causes a rapidly progressive glomerulonephritis. The pathogenic antibody response is directed to the non-collagenous (NC1) domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1), but not to the homologous region of the alpha1(IV)NC1. To identify the conformation-dependent immunodominant epitope on the alpha3(IV)NC1, a variety of recombinant NC1 domains were constructed by replacing single residues of alpha3(IV) with the corresponding amino acids from the nonreactive alpha1(IV) chain. Replacement mutations were identified that completely destroyed the Goodpasture epitope in the alpha3(IV) chain. Based on the identification of these critical positions, the epitope was finally reconstructed within the frame of the alpha1(IV) chain. The substitution of nine discontinuous positions in the alpha1(IV)NC1 with amino acid residues from the alpha3 chain resulted in a recombinant construct that was recognized by all patients' sera (n = 20) but by none of the sera from healthy controls (n = 10). This provides, for the first time, the molecular characterization of a single immunodominant conformational epitope recognized by pathogenic autoantibodies in a human autoimmune disease, representing the basis for the development of new epitope-specific strategies in the treatment of Goodpasture disease

The prognostic significance in Goodpasture's disease of specificity, titre and affinity of anti-glomerular-basement-membrane antibodies.

Background: The nephrotoxic potential of anti-glomerular-basement-membrane (GBM) antibodies has been demonstrated in numerous animal experiments. However, it is not known to what extent the properties of circulating anti-GBM antibodies in human disease reflect the severity of the disease and predict the outcome. Methods: Clinical data were collected for 79 Swedish patients for whom a positive result had previously been obtained with anti-GBM ELISA. In stored sera from the patients, we measured antibody concentration, specificity and affinity together with antineutrophil cytoplasmic antibodies and alpha(1)-antitrypsin phenotype. Results: Six months after diagnosis, 27 (34%) were dead, 32 (41%) were on dialysis treatment and only 20 (25%) were alive with a functioning native kidney. The best predictor for renal survival was renal function at diagnosis. In patients who were not dialysis dependent at diagnosis however, renal survival was associated with a lower concentration of anti-GBM antibodies, a lower proportion of antibodies specific for the immunodominant epitope and the histological severity of the renal lesion. The only factor that correlated with patient survival was age. Conclusions: Immunochemical properties of autoantibodies do not affect patient survival in anti-GBM disease but seem to be a factor in renal survival in patients detected before renal damage is too advanced. Copyright (C) 2003 S. Karger AG, Basel

Molecular properties of the Goodpasture epitope

Goodpasture disease fulfils all criteria for a classical autoimmune disease, where autoantibodies targeted against the non-collagenous domain of the alpha3-chain of collagen IV initiates an inflammatory destruction of the basement membrane in kidney glomeruli and lung alveoli. This leads to a rapidly progressive glomerulonephritis and severe pulmonary hemorrhage. Previous studies have indicated a limited epitope for the toxic antibodies in the N-terminal part of the non-collagenous domain. The epitope has been partially characterized by recreating the epitope in the non-reactive alpha1-chain by exchanging nine residues to the corresponding ones of alpha3. In this study we have investigated to what extent each of these amino acids contribute to the antibody binding in different patient sera. The results show that seven of the nine substitutions are enough to get an epitope that is recognized equally well as the native alpha3-chain by all sera from 20 clinically verified Goodpasture patients. Furthermore, the patient sera reactivity against the different recombinant chains used in the study are very similar, with some minor exceptions, strongly supporting a highly defined and restricted epitope. We are convinced that the restriction of the epitope is of significant importance for the understanding of the etiology of the disease. Thereby also making every step on the way to characterization of the epitope a crucial step on the way to specific therapy for the disease

Characterization of anti-GBM antibodies involved in Goodpasture's syndrome

Characterization of anti-GBM antibodies involved in Goodpasture's syndrome. Goodpasture's syndrome is a life threatening autoimmune kidney disease. The patients have autoantibodies to the glomerular basement membrane, which are specific for the C-terminal domain of type IV collagen (NC1). The major antigen has been localized to the alpha3(IV)-chain. We have investigated sera from 44 patients with anti-NC1 antibodies. The quantity of antibodies to four different alpha(IV)-chains of type IV collagen was measured with direct ELISA. We used affinity chromatography to separate the antibodies and their specificities were studied with ELISA. The results show that about 1% of the patients total IgG are anti-NC1 antibodies and that 90% of these antibodies are specific for the alpha3(IV)-chain. Antibodies to the other alpha(IV)-chains were found in 80% of the patients. Furthermore, affinity purified anti-alpha3(IV) antibodies from one patient were inhibited by antibodies from the other patients, from 4 to 72%. The antibodies, from 39 of the patients, were inhibited by a monoclonal antibody against the alpha3(IV)-chain. The results indicate that patients with Goodpasture's syndrome can have antibodies to most of the alpha(IV)-chains, while the majority of anti-NC1 antibodies are restricted to the alpha3(IV)-chain. Moreover the number of epitopes seems to be limited and the majority of the antibodies from most patients are against one single epitope on the alpha3(IV)-chain