PATIENTS AND CONTROLS

Sera from 18 UC patients were selected from a serum bank fulfilling the following criteria: positive for IgG-ANCA with a titre ⩾1/320; a clear ANCA perinuclear pattern; and negative for antinuclear antibodies (ANA) tested with HEp-2 cells and rat liver sections using an indirect immunofluorescence (IIF) assay. A diagnosis of UC was established using the Lennard-Jones clinicopathological criteria.9 Seven patients had active and two inactive disease, as assessed by the Truelove index.10 The remaining nine patients had been colectomised at least two years before sampling. Four had total proctocolectomy with Brooke's ileostomy, and an ileal pouch anal anastomosis had been performed in five. Serum samples were stored at −70°C until analysis. Sera from three healthy individuals, negative for both ANCA and ANA, were used as controls.

POLYMORPHONUCLEAR LEUCOCYTE ISOLATION

Polymorphonuclear leucocytes (PMN) were isolated from peripheral blood of healthy volunteers using a standard technique with a solution containing sodium metrizoate and dextran 500 as previously described.11 The final suspension contained more than 95% PMN, as counted on a haemocytometer.

PMN CULTURE AND INDUCTION OF APOPTOSIS

Freshly isolated PMN (2.5×10⁶ cells/ml) were cultured and incubated for 18 hours at 37°C in a humidified atmosphere containing 5% CO₂ in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum and antibiotics. Cycloheximide (10 μg/ml) was added to the medium to induce increased apoptosis.12 ,13 Apoptosis was demonstrated using three well established methods: (1) TUNEL [TdT mediated fluorescein nucleotides (FITC-dUTP)nick endlabelling] (Boehringer Manheim, cat. No 1684 795), an in situ programmed cell death labelling method based on detection of DNA strand breaks labelling free 3′-OH termini with FITC-dUTP. Terminal deoxynucleotydil transferase (TdT) is the enzyme which catalyses polymerisation of modified nucleotides to free 3′-OH DNA ends. Incorporated fluorescein labelled dUTP was analysed under fluorescence and confocal laser scanning microscopy14; (2) detection of chromatin fragmentation into histone associated DNA fragments (mono- and oligonucleosomes). This gives a distinctive DNA ladder pattern on purification of lysed cells in 1% agarose gel electrophoresis15; and (3) analysis of cell morphology of cytocentrifuged preparations using methyl blue staining.15

ANCA DETECTION

Glass slides containing cytocentrifuged smears (Shandon Cytospin, Shandon Inc., Pittsburg, Pennsylvania, USA) of non-apoptotic and apoptotic human neutrophils from healthy volunteers (approximately 100 000 per slide) were fixed in 100% ethanol at room temperature for five minutes and used as substrate. Fixed neutrophils were stored at −20°C until use. Sera from UC ANCA positive patients and healthy controls were tested at 1/40 dilution. Serum IgG-ANCA were visualised with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) conjugated rabbit antibodies against human IgG (γ chains) (DAKO AS, F202, and R151, respectively) diluted 1/100, and examined under fluorescence microscopy and confocal laser scanning microscopy (see below).

NON-APOPTOTIC DNA DETECTION

Propidium iodide (PI) staining (5 μg/ml; 10 minutes; Sigma, St Louis, Missouri, USA) was used in ethanol fixed neutrophil slides to visualise nuclear DNA.

APOPTOTIC DNA DETECTION

The TUNEL technique was used to detect DNA breaks or nicks with labelled FITC-dUTP in slides containing apoptotic neutrophils (see above).

INDIRECT IMMUNOFLUORESCENCE MICROSCOPY

Slides containing non-apoptotic or apoptotic neutrophils were used. FITC conjugated rabbit antibodies against human IgG were used to visualise serum IgG-ANCA under fluorescence microscopy at 400× (Zeiss, Axioplan, Germany). Slides were assessed by two independent observers who were unaware of the patient's diagnosis. Control sera positive and negative for ANCA were included in each test batch. For each serum sample, ANCA titres using non-apoptotic and apoptotic neutrophils were quantified.

CONFOCAL LASER SCANNING MICROSCOPY

To assess possible colocalisation of nuclear DNA/apoptotic DNA and ANCA, slides were examined after double immunofluorescence using confocal laser scanning microscopy. Confocal images were obtained with a Leica TCS NT scanning module (Leica Microsystems Heidelberg GmbH, Germany) with a Kripton-Argon laser, coupled to a Leica DMRB microscope with 100× objective and using a 488 nm laser line and a 530/30 band pass filter for the FITC signal (channel 1) and 568 nm laser line and 590 long pass filter for the PI/TRITC signal (channel 2). Optical sectioning of neutrophils was performed in 500 nm steps and overlay images were recorded superimposing simultaneous images from each channel. Double staining was performed for ANCA (FITC) and non-apoptotic DNA (PI), and for ANCA (TRITC) and apoptotic DNA (FITC).

Colocalisation was identified in the overlay images by yellow staining caused by the mixture of the green signal from FITC and the red signal from TRITC or PI as follows: (1) colocalisation in non-apoptotic neutrophils was the result of superimposition of the green fluorescence of FITC labelled secondary antibodies which detect ANCA and the red signal of PI which stains DNA; (2) colocalisation in apoptotic neutrophils was the result of superimposition of the green fluorescence of FITC-TUNEL which detects apoptotic DNA and the red signal of TRITC labelled secondary antibodies which detect ANCA. Yellow staining is more evident in those areas with similar immunofluorescence intensity for the two channels. In some areas of Ag-ANCA colocalisation with different immunoflorescence intensities, the red or green colour may predominate. Thus to give a more exact pattern of Ag-ANCA location, avoiding neutrophil image interpretation, colocalisation was also demonstrated graphically by a profile of fluorescence intensity obtained for each channel in a single line drawn through the cell sections.

CONVENTIONAL INDIRECT IMMUNOFLUORESCENT ASSAY

On IIF examination with cytocentrifuged ethanol fixed non-apoptotic neutrophils, seven sera had an ANCA titre of 1/1280, six had a titre of 1/640, and five a titre of 1/320. When retested using cytocentrifuged ethanol fixed apoptotic neutrophils, most sera also showed intense fluorescence with no significant changes with respect to the titres obtained with non-apoptotic neutrophils. The titres of only three sera decreased by two dilutions compared with the titre found with non-apoptotic neutrophils. In no case did an increase occur in ANCA titres observed using apoptotic neutrophils.

INDIRECT IMMUNOFLUORESCENT LASER CONFOCAL MICROSCOPY

Non-apoptotic neutrophils

For cellular UC-ANCA distribution, three patterns of confocal laser immufluorescence were observed: (1) diffuse nuclear localisation, as illustrated by sera from three patients (16.7%). In these cases yellow staining, corresponding to the overlap of the green ANCA fluorescence and the red DNA fluorescence, was observed in the whole nuclear area. The profile of the staining intensity obtained by channel 1 (green profile, ANCA) completely overlapped with that obtained by channel 2 (red profile, DNA) (fig 1A). In addition, the intensity of the ANCA fluorescence profile remained high in the entire area bounded by the DNA fluorescence profile; (2) peripheral nuclear localisation, as illustrated by sera from nine patients (50%). In this pattern a yellow rim-like staining corresponding to the green ANCA fluorescence in the nuclear periphery overlapping with the red DNA fluorescence was observed. The profile obtained by ANCA staining (green profile) was mainly localised in the periphery of the nucleus as seen by two peaks of greater intensity of ANCA staining in this location (fig 1B); and (3) mixed nuclear and cytoplasmic localisation, as illustrated by the remaining sera from six subjects (33.4%) (fig 1C). In these cases the ANCA fluorescence profile (green profile) was partly superimposed on the DNA profile (red profile) but mainly exceeded the DNA boundary.

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Figure 1

Confocal laser scanning microscopy view of non-apoptotic ethanol fixed neutrophils. Antineutrophil cytoplasmic antibodies (ANCA) were detected with fluorescein isothiocyanate (FITC) conjugated secondary antibodies that produce a green signal at a wavelength of 500–560 nm. DNA was visualised with propidium iodide which gives a red signal at wavelengths longer than 590 nm. Three different patterns of ANCA localisation may be observed. (A) A diffuse nuclear ANCA associated ulcerative colitis (UC-ANCA) staining pattern, as demonstrated by yellow staining in the whole nuclear area as a result of superimposition of the two fluorescence signals. Complete overlay of the profile of staining intensity obtained by channel 1 (green profile, ANCA) and that obtained by channel 2 (red profile, DNA) may be observed. (B) Peripheral nuclear staining pattern, as demonstrated by a yellow rim-like ANCA fluorescence in the inner side of the nuclear periphery, overlapping with DNA. Yellow staining may be observed in the peripheral nuclear area as a result of superimposition of the two fluorescence signals. The profile obtained by channel 1 (green profile, ANCA) mainly localises in the inner side of the nucleus (channel 2, red profile, DNA) periphery, as demonstrated by two peaks of greater intensity of ANCA staining in this location. (C) Mixed nuclear and cytoplasmic staining pattern. The green staining of the fluorescence signal of ANCA, non-superimposed with DNA, may be observed. The profile obtained by channel 1 (green profile, ANCA) is partly localised in the nucleus (channel 2, red profile, DNA) but overwhelms the DNA fluorescence boundary. Bar=10 μm.

Apoptotic neutrophils

In all sera, ANCA fluorescence almost completely colocalised with apoptotic DNA and was observed for all three types of ANCA patterns. This may be seen as diffuse yellow staining as a result of ANCA rhodamine labelling and TUNEL fluorescein labelling superimposition (fig 2A). The ANCA profile (red profile) almost completely overlapped with the cleaved DNA profile (green profile). Only in three sera did some areas of the neutrophil show ANCA staining intensity higher than that of the apoptotic DNA. These areas seemed to correspond to apoptotic blebs which contained cleaved proteins and DNA. This occurred in some, but not all, neutrophils of the same slides (fig2B).

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Figure 2

Confocal laser scanning microscopy of apoptotic ethanol fixed neutrophils. Antineutrophil cytoplasmic antibodies (ANCA) were detected with tetramethylrhodamine isothiocyanate (TRITC) which produces a red signal at wavelengths longer than 590 nm. Apoptotic DNA was visualised with TUNEL (TdT mediated FITC-dUTP nick end labelling), an in situ programmed cell death labelling method based on detection of DNA strand breaks labelled with fluorescein isothiocyanate (FITC) which gives a green signal at wavelengths of 500–560 nm. (A) With most sera, ANCA staining showed diffuse colocalisation with apoptotic cleaved DNA (oligonucleosomes and nucleosomes). Cross over fluorescence between FITC (cleaved DNA) and TRITC (ANCA) is shown by yellow staining. Almost complete superimposition of the profile of staining intensity obtained by channel 1 (red profile, ANCA) and that obtained by channel 2 (green profile, cleaved DNA) may be observed. (B) With three sera the ANCA staining intensity (red signal) was higher than that of apoptotic DNA (green signal) in some areas of the apoptotic neutrophils. These areas morphologically correspond to apoptotic blebs beyond the cytoplasmic membrane. The ANCA fluorescence profile (green profile) partly superimposed with the DNA profile (red profile) but exceeded the boundary of the apoptotic DNA. Bar=10 μm.