Apoptotic Cell-Induced, Antigen-Specific Immunoregulation to Treat Experimental Antimyeloperoxidase GN.

BACKGROUND: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. METHODS: To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. RESULTS: MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. CONCLUSIONS: These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.

Immunoglobulins G from patients with ANCA-associated vasculitis are atypically glycosylated in both the Fc and Fab regions and the relation to disease activity.

BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity. METHODOLOGY: Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments. PRINCIPAL FINDINGS: IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion. CONCLUSIONS/SIGNIFICANCE: Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases.

ANCA-Associated Glomerulonephritis: Risk Factors for Renal Relapse.

Relapse in ANCA-associated vasculitis (AAV) has been studied previously, but there are few studies on renal relapse in particular. Identifying patients at high risk of renal relapse may aid in optimizing clinical management. We investigated which clinical and histological parameters are risk factors for renal relapse in ANCA-associated glomerulonephritis (AAGN). Patients (n = 174) were newly diagnosed and had mild-moderate or severe renal involvement. Data were derived from two trials of the European Vasculitis Society: MEPEX and CYCAZAREM. The Cox regression model was used to identify parameters increasing the instantaneous risk (= rate) of renal relapse (useful for instant clinical decisions). For identifying predictors of renal relapse during follow-up, we used Fine & Gray's regression model. Competing events were end-stage renal failure and death. The cumulative incidence of renal relapse at 5 years was 9.5% (95% CI: 4.8-14.3%). In the Cox model, sclerotic class AAGN increased the instantaneous risk of renal relapse. In Fine & Gray's model, the absence of interstitial infiltrates at diagnosis was predictive for renal relapse. In this study we used two different models to identify possible relationships between clinical and histopathological parameters at time of diagnosis of AAV with the risk of experiencing renal relapse. Sclerotic class AAGN increased the instantaneous risk of renal relapse. This association is most likely due to the high proportion of sclerosed glomeruli reducing the compensatory capacity. The absence of interstitial infiltrates increased the risk of renal relapse which is a warning sign that patients with a relatively benign onset of disease may also be prone to renal relapse. Renal relapses occurring in patients with sclerotic class AAGN and renal relapses occurring in patients without interstitial infiltrates were mutually exclusive, which may indicate that they are essentially different.

Antineutrophil cytoplasmic autoantibodies: how are they detected and what is their use for diagnosis, classification and follow-up?

Antineutrophil cytoplasmic antibodies (ANCA) are traditionally detected by an indirect immunofluorescence technique. According to the international consensus on ANCA testing, ANCA should also be tested by antigen-specific tests for myeloperoxidase-ANCA and proteinase 3-ANCA. The direct noncompetitive enzyme-linked immunosorbent assay (ELISA) used to be the method of choice. Nowadays, these assays are called "first-generation" assays. Second-generation tests (capture ELISA) or third-generation tests (anchor ELISA) are more sensitive and specific for ANCA testing. We postulate that ANCA as detected by these newer ANCA tests may replace the need to perform indirect immunofluorescence-based assays. For classification of patients, ANCA serotype seems more important than classifying patients according to their clinical subtype, since genetics, clinical manifestations and response to therapy are more related to ANCA serotype than to clinical subtype. Detection of ANCA to monitor disease activity is still a controversial issue. Treatment based on ANCA levels is at present only experimentally performed in those patients who are treated with B-cell depletion therapy with rituximab. Future studies are needed to establish whether this way of monitoring patients is warranted.

Influence of variable domain glycosylation on anti-neutrophil cytoplasmic autoantibodies and anti-glomerular basement membrane autoantibodies.

BACKGROUND: The pathophysiological significance of variable region glycosylation of autoantibodies is still unclear. In the current study, the influence of the variable region N-linked oligosaccharides on the reactivity of three autoantibody specificities was investigated with Sambucus nigra agglutinin (SNA), which mainly binds to oligosaccharides with terminal α2, 6-linked sialic acid on the variable region of IgG. METHODS: Twenty-seven patients with serum positive anti-neutrophil cytoplasmic autoantibodies (ANCA) against myeploperoxidase (MPO) or proteinase 3 (PR3), or autoantibodies against glomerular basement membrane (GBM) were included. Total IgG was isolated and separated into non-SNA-binding and SNA-binding fractions with SNA affinity chromatography. Antigen-specific IgG was purified by immunoaffinity chromatography. RESULTS: At the same concentration of IgG, the antigen binding level of non-SNA-binding IgG was significantly lower than that of SNA-binding IgG for MPO-ANCA (absorbance value at 405 nm, 0.572 ± 0.590 vs. 0.962 ± 0.670, P < 0.001) and for PR3-ANCA (0.362 ± 0.530 vs. 0.560 ± 0.531, P = 0.003). The antigen binding level of non-SNA-binding IgG was significantly higher than that of SNA-binding IgG for anti-GBM antibodies (1.301 ± 0.594 vs. 1.172 ± 0.583, P = 0.044). The level of variable region glycosylation of total IgG was significantly lower than that of affinity-purified MPO-ANCA (1.021 ± 0.201 vs. 1.434 ± 0.134, P = 0.004). The level of variable region glycosylation of total IgG was significantly higher than that of affinity-purified anti-GBM antibodies (1.034 ± 0.340 vs. 0.734 ± 0.333, P = 0.007). The SNA-binding fraction of MPO-ANCA-containing IgG and PR3-ANCA-containing IgG induced higher levels of neutrophil oxygen radical production than the corresponding non-SNA-binding fractions (P < 0.001 and P = 0.043, respectively). The level of variable region glycosylation of affinity-purified MPO-ANCA was higher in active AAV than the same patients in remission (P = 0.001). CONCLUSION: Characteristics of variable region glycosylation of ANCA and anti-GBM antibodies were different from that of total IgG, which might influence the antigen-binding ability of these antibodies. Variable region glycosylation of ANCA might influence the effect of ANCA-induced neutrophils respiratory burst.

Conventional apheresis therapies: a review.

This article reviews advances in the scientific basis and medical practice of plasmapheresis and cytapheresis therapies. Newly-characterized autoantibodies in neuromyelitis optica, Guillain-Barre variants, anti-neutrophil cytoplasmic antibody (ANCA) vasculitides, etc., exemplify the modern molecular biology which now provides a rigorous framework of understanding for the clinical practice of plasmapheresis. Clinical trials continue to clarify the appropriate use of therapeutic plasmapheresis (TPE) in these and other diseases. Centrifugal (cTPE) and membrane filtration (mTPE) types of plasmapheresis are compared, with details of the plasmapheresis prescription, anticoagulation choices, replacement fluids and other practical considerations. Plasma removal is more efficient with cTPE; mTPE systems have a lower plasma extraction ratio, and therefore require higher blood flow rates or longer procedure times. Autoantibodies and other pathogenic macromolecules targeted for removal by plasmapheresis can be depleted predictably when the plasma is discarded, as in conventional TPE. On-line plasma processing to regenerate the patient's own plasma avoids the need for replacement albumin solutions or plasma transfusion, but is inherently less efficient at removing the target molecule, so usually requires a longer procedure. Therapeutic white cell reduction (leukapheresis), platelet reduction (thrombocytapheresis) and red cell exchange (erythrocytapheresis) require centrifugal apheresis systems.

Prevalence and clinical significance of auto-antibodies in adults with cystic fibrosis.

The aim of this study was to determine the prevalence of different auto-antibodies in adult, French cystic fibrosis (CF) patients and to look for a correlation between autoimmunity, patient characteristics and survival. The sera of 144 patients were screened for a wide range of antibodies. Clinical, biological and bacteriological characteristics and the cystic fibrosis transmembrane conductance regulator genotype were recorded and progression of lung disease was examined. 113 (78.5%) patients displayed one or several auto-antibodies, predominantly immunoglobulin (Ig)A anti-Saccharomyces cerevisiae antibodies (ASCA; 43.7%) and antineutrophil cytoplasmic antibodies (ANCA; 40%), of which 59% showed bactericidal/permeability-increasing protein (BPI) specificity. The presence of BPI-ANCA was associated with the number of antibiotic courses, low body mass index, Pseudomonas aeruginosa colonisation, the presence of resistant P. aeruginosa, low forced expiratory volume in 1 s, CF-related liver disease, hypergammaglobulinaemia, male sex and inflammatory syndrome. The presence of ASCA-IgA was correlated with male sex and hypergammaglobulinaemia. 41 patients presented with chronic respiratory failure and/or requested lung transplantation or died during follow-up. These events were more frequent in patients with BPI-ANCA or ASCA-IgA. These findings confirm the high frequency of auto-antibodies in CF, particularly BPI-ANCA and ASCA-IgA, and the link between BPI-ANCA, severity of lung disease and CF prognosis.

Rapid decrease of anti-beta-glucan antibody as an indicator for early diagnosis of carinii pneumonitis and deep mycotic infections following immunosuppressive therapy in antineutrophil cytoplasmic antibody-associated vasculitis.

Deep mycosis (aspergillus pneumonia (AsP)) and carinii pneumonitis (PCP) are complications of immunosuppressive treatment for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The objective was to clarify the clinical significance of plasma titer of antibody against beta-glucans (anti-BG antibody) as a predictor of complications such as AsP or PCP and the prognosis of patients. Enzyme-linked immunosorbent assay was used to measure the plasma titer of antibodies against beta-glucans (BG) from Candida albicans in 22 healthy subjects and 52 patients with various stages of AAV. The mean plasma titer of the anti-BG antibody was 2,677 +/- 1,686 U in healthy subjects, 691 +/- 522 U in patients with untreated active vasculitis (n = 14), and 547 +/- 416 U in patients soon after immunosuppressive treatment (n = 24). Healthy subjects had significantly higher antibody titers than the other two groups (P < 0.05). Repeated measurements over the clinical course of AAV revealed an increase during remission to 1,180 +/- 130 U (n = 11), while there was a significant rapid decrease to 369 +/- 441 U (P < 0.01) concomitantly with elevation in plasma C-reactive protein and BG levels in patients with AAV that had AsP or PCP infection. Antifungal therapy resulted in a rapid rise of anti-BG antibody titer. Experiments in mice suggested that the anti-BG antibody neutralizes BG. Rapid decrease of the anti-BG antibody titer may be a useful indicator for diagnosis of the presence of AsP or PCP and for estimating the prognosis of patients with these opportunistic infections during immunosuppressive treatment of AAV.

Testing on formalin-fixed neutrophils is less sensitive and specific for small vessel vasculitis, and less sensitive for MPO-ANCA, than most ELISAs.

This study evaluated the usefulness of testing sera with perinuclear antineutrophil cytoplasmic antibodies (P-ANCA) on formalin-fixed neutrophils to differentiate between vasculitis and other diseases, and to distinguish between myeloperoxidase (MPO) and other antigen targets. Sera from active (n=24) or treated vasculitis (n=23) and non-vasculitic disease (n=37) were tested in 3 ethanol- and 2 formalin-fixed neutrophil assays, and in 12 MPO-ANCA ELISAs. The sensitivity of demonstrating that P-ANCA became cytoplasmic on formalin-fixed cells in vasculitis, and negative in non-vasculitic disease was 75-79% in different assays compared with 88% for positivity in the majority of MPO-ANCA ELISAs. The sensitivity and specificity of demonstrating that P-ANCA became cytoplasmic where the target was MPO, and disappeared with other antigens were 89% and 74-80% respectively in different assays. P-ANCA specificity for MPO should be confirmed in one of the better-performing MPO-ANCA ELISAs.

A study of conformational restraints on reactivity of human PR3-specific autoantibodies (ANCA) facilitated through protein folding manipulations of a new recombinant proteinase 3 protein.

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3(+his), EK-cleaved (his-tag removed) rPR3(- his), and EK-cleaved, denatured/refolded rPR3(- his/dr) (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3(- his/dr) than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.

Renal histology in Chinese patients with anti-myeloperoxidase autoantibody-positive Wegener's granulomatosis.

BACKGROUND: Proteinase-3 antineutrophil cytoplasmic antibody (PR3-ANCA) was the serological marker for Wegener's granulomatosis (WG), while myeloperoxidase (MPO)-ANCA was the serological marker for microscopic polyangiitis (MPA). However, our previous study suggested that patients with MPO-ANCA positive WG were common in Chinese. This study aimed to analyse the renal histology of patients with MPO-ANCA positive WG. METHODS: Patients in our centre with WG were selected according to both the Chapel Hill Consensus Conference (CHCC) definition and American College of Rheumatology classification criteria. Patients with MPA were selected according to the CHCC definition. The renal histology was compared between patients with MPO-ANCA positive WG and with PR3-ANCA positive WG as well as patients with MPO-ANCA positive MPA. RESULTS: Sixty-one patients with WG had complete renal histological data, 39/61 with positive MPO-ANCA and 22/61 with positive PR3-ANCA. Among patients with crescents in glomeruli, those with MPO-ANCA had fewer cellular crescents and more fibrous crescents than those with PR3-ANCA (P < 0.01 and P < 0.05, respectively). Interstitial fibrosis and tubular atrophy were more prevalent and severe in patients with MPO-ANCA than in those with PR3-ANCA (P < 0.01 and P < 0.05, respectively). Compared with 44 patients with MPO-ANCA positive MPA, patients with MPO-ANCA positive WG had fewer glomeruli with crescents and more normal glomeruli (P < 0.01 and P < 0.01, respectively). CONCLUSION: Patients with MPO-ANCA positive WG are common in Chinese. In renal histology, chronic lesions were more severe and prevalent in patients with MPO-ANCA positive WG than in patients with PR3-ANCA positive WG. Glomerular lesions were less severe and less prevalent in patients with MPO-ANCA positive WG than in those with MPO-ANCA positive MPA.

Nanocrystal quantum dot-conjugated anti-myeloperoxidase antibody as the detector of activated neutrophils.

Fluorescent nanocrystal quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. Here we show that QDs conjugated with antibody against neutrophil peroxidase, myeloperoxidase (MPO). We designed a novel method to conjugate QDs to antibody without losing any antibody function including their antigen recognizing and Fc-receptor binding activities. When we applied anti-MPO antibody (Ab) with conventional organic probes in the case of immunostaining of living cells, the antibodies lost their fluorescence because of MPO enzymic activity to produce reactive oxygen species. Our QD-conjugated anti-MPO (alpha-MPO-QDs) can detect MPO on the surface of activated neutrophils. In addition, anti-MPO-QDs did not react to the inactivated neutrophils. In conclusion, we demonstrated that antibody visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. Taken together, these techniques have the possibility that QDs can reveal the activation of neutrophils by immunostaining and flow cytometric analysis as a powerful tool for diagnosis of the neutrophil activation in vitro.

Persistent T-cell activation and clinical correlations in patients with ANCA-associated systemic vasculitis.

BACKGROUND: Although in antineutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AASV) patients, activation of T-cells has been described, persistence of these alterations has not been well characterized. This study was conducted to define persistent T-cell activation (PTA) in AASV patients and to assess whether this correlates with disease activity, disease severity, age or therapy. METHODS: The expression of CD4, CD45RO, CD25, CD26, CD28, CCR7 and HLA-DR was examined longitudinally in 38 consecutive AASV patients. Clinical parameters were compared by univariate and multiple analysis and Kaplan-Meier curves for relapse-free survival were calculated. RESULTS: PTA could be defined as either of two activation phenotypes, i.e. a low percentage of CD4+ CD45RO- T-cells or a high percentage of CD25 in the naïve CD4+ population (n = 26), since only these phenotypes were stable over time and were not associated with active disease. In patients with PTA, major organ involvement was significantly more often found than in patients without PTA. Moreover, the cumulative cyclophosphamide dose (26.86 vs 8.53 P < 0.01) was significantly increased in these patients, suggesting that PTA was associated with disease severity. In general, patients with PTA were older than those without (62.92 +/- 9.4 years vs 48.42 +/- 16.9 years respectively, P < 0.01). PTA was independent of disease duration. Interestingly, patients with a low percentage of CD4+CD45RO- T-cells were significantly more often diagnosed as microscopic polyangiitis (P < 0.01). CONCLUSION: We identified two independent phenotypes of T-cell activation in AASV patients. These phenotypes are persistent and do not reflect disease activity. PTA predominantly occurs in patients with severe disease. This might explain the higher cumulative cyclophosphamide dose found in these patients.

The importance of resolution of inflammation in the pathogenesis of ANCA-associated vasculitis.

The primary small-vessel systemic vasculitides are disorders that target small blood vessels, inducing vessel wall inflammation, and are associated with the development of anti-neutrophil cytoplasmic antibodies. Multiple organs are attacked, including the lungs and kidneys. Increasing knowledge of pathogenesis suggests that the antibodies activate neutrophils inappropriately, leading to endothelial and vascular damage. Cytokines, such as tumour necrosis factor, can facilitate damage by priming the neutrophils and activating endothelial cells. Apoptosis of infiltrating neutrophils is also disrupted by anti-neutrophil cytoplasmic antibody activation, and removal of these effete cells occurs in a pro-inflammatory manner, promoting persistent inflammation. The autoimmune response may be promoted by aberrant phagocytosis of apoptotic neutrophils by dendritic cells. Understanding the pathogenesis can help to rationalize existing therapies and indicate new approaches to therapy.

Wegener's granulomatosis--autoimmunity to neutrophil proteinase 3.

Wegener's granulomatosis (WG) is a small-vessel vasculitis associated with various clinical manifestations, among which the most common are respiratory tract disease and glomerulonephritis leading to renal failure. The pathogenesis of vascular injury in WG is ascribed to antineutrophil cytoplasmic antibodies (ANCA) directed mainly against proteinase 3 (PR3), an enzyme from neutrophil granules. The reasons for the breakdown of self tolerance to PR3 are unknown, and together with the molecular mechanisms underlying this immunoinflammation, are the subject of research. Standard treatment of WG consists of cyclophosphamide and corticosteroids. In patients resistant to this therapy or with refractory disease, some alternative strategies involving tumor necrosis factor blockade, polyclonal antithymocyte globulin or monoclonal anti-T cell antibodies are applied.

Amphipathic variable region heavy chain peptides derived from monoclonal human Wegener's anti-PR3 antibodies stimulate lymphocytes from patients with Wegener's granulomatosis and microscopic polyangiitis.

Amphipathic variable-region heavy chain 11-mer peptides from monoclonal human IgM antiproteinase-3 antibodies were studied for peripheral blood lymphocyte stimulation in 21 patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA), connective tissue disease controls and normal control subjects. Positive T-cell activation was observed in most experiments with WG patients' lymphocytes using amphipathic VH-region peptides from four different human monoclonal anti-PR3 antibodies. Control peptides of the same length but without amphipathic characteristics along with other amphipathic peptides not derived from monoclonal anti-PR3 sequence were employed as controls. No significant lymphocyte stimulation was observed with normal controls, but positive stimulation with amphipathic VH peptides was also recorded in other connective tissue disease controls mainly patients with rheumatoid arthritis. Amphipathic peptides not derived from anti-PR3 sequence did not stimulate WG lymphocytes. Our findings indicate that lymphocyte reactivity as an element of cell-mediated immunity may be activated by amphipathic VH-region amino acid sequences of autoantibodies which are themselves associated with diseases such as WG.

Testing for antineutrophil cytoplasmic antibodies.

The most common reason to request a test for antineutrophil cytoplasmic antibodies (ANCA) is to diagnose Wegener's granulomatosis and microscopic polyangiitis and to monitor inflammatory activity in these diseases. Several retrospective and prospective studies have suggested that the demonstration of ANCA lacks sensitivity and specificity, but these series have detected ANCA with neutrophil-indirect immunofluorescence alone, have used a disease classification that did not describe microscopic polyangiitis and have included patients with inactive disease. The 'International Consensus Statement on Testing and Reporting ANCA' has been developed to optimize the clinical relevance of ANCA testing by the adoption of standardized testing and reporting procedures. International collaborative efforts continue to focus on improving the tests for ANCA.

Heat treatment of normal human sera reveals antibodies to bactericidal permeability-inducing protein (BPI).

Heat treatment of normal sera to 56 degrees C for 30 min, a common procedure for the inactivation of viruses, e.g. HIV, reveals the presence of antibodies to neutrophil cytoplasm antigens (ANCA), as detected by indirect immunofluorescence on ethanol-fixed human neutrophils and by antigen-specific ELISA for BPI. Reactivity was not seen to the other common vasculitis-associated antigens proteinase 3 (PR3) or myeloperoxidase (MPO). The effect of temperature was maximal at 56 degrees C, with substantial antibody demonstrable after only 5 min at this temperature. In experiments using polyethylene glycol (PEG)6000 to remove immune complexes, the effect of heating could be abrogated by preincubation with 8% PEG, which suggested that these anti BPI antibodies might be complexed in sera. After passage of normal plasma over a protein G column, the acid-eluted fraction contained elevated levels of antibodies to BPI but not to other vasculitis-associated antigens such as PR3 or MPO, nor to glomerular basement membrane (GBM), the Goodpasture antigen which is recognized by the pathogenically important human antibodies shown to mediate nephritis in transfer experiments. Moreover the levels of anti-BPI in the IgG fraction could be augmented by preincubation with glycine pH 2.5 for 30 min. This anti-BPI activity could be inhibited by addition of the unbound material from the protein G column and this inhibitory material was not heat-labile at 56 degrees C. The molecular specificity of this autoreactivity was confirmed using recombinant BPI in coincubation experiments and the epitope localized to the C or N terminal moieties by the use of recombinant fusion proteins.