Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-ANCA) using immunofluorescence microscopy

Abstract

Perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) directed against cytoplasmic proteins of neutrophils have been studied extensively in patients with systemic vasculitides. Recent data indicate that antineutrophil antibodies in sera from patients with chronic inflammatory bowel diseases (IBD) or autoimmune liver disorders, currently called 'atypical p-ANCA', recognize a nuclear target antigen, rendering the term 'ANCA' inaccurate. Specific microscopic criteria to distinguish atypical p-ANCA from p-ANCA are lacking. We used planar and confocal laser scanning indirect immunofluorescence microscopy to examine the labelling characteristics of ethanol-, methanol- and formaldehyde-fixed neutrophils by antineutrophil antibodies in 153 serum samples from patients with IBD, autoimmune liver disorders, systemic vasculitides or healthy blood donors. On ethanol- or methanol-fixed neutrophils, multiple intranuclear fluorescent foci together with either a rim-like peripheral nuclear staining ('type A') or a combined cytoplasmic and peripheral nuclear staining ('type B') was noted exclusively with atypical p-ANCA in sera from patients with IBD or autoimmune liver disorders. Intranuclear foci, which probably corresponded to invaginations of the nuclear envelope, were not labelled by p-ANCA from patients with microscopic polyangiitis or cytoplasmic ANCA (c-ANCA) from patients with Wegener's granulomatosis. On formaldehyde-fixed neutrophils, atypical p-ANCA gave a fine rim-like staining of the nuclear periphery, whereas ANCA diffusely labelled the cytoplasm. To distinguish reliably between the patterns produced by atypical p-ANCA or p-ANCA, particularly p-ANCA, careful indirect immunofluorescence microscopy on ethanol- as well as on formaldehyde-fixed neutrophils is necessary, with particular emphasis on the presence of multiple intranuclear fluorescent foci.

Fig. 1

Immunofluorescence micrographs of staining patterns produced by different types of antineutrophil antibodies on ethanol-fixed human neutrophils examined by planar and confocal laser scanning indirect immunofluorescence microscopy. Labelling of ethanol-fixed neutrophils by antineutrophil antibodies was detected with FITC-conjugated goat antihuman IgG secondary antibodies. The micrographs in panels a, c, e and g have been visualized by indirect planar immunofluorescence microscopy, the images in panels b, d, f and h were taken by confocal laser scanning microscopy. (a and b) A diffuse cytoplasmic fluorescence, frequently accentuated between the nuclear lobes of the neutrophils, was characteristic of a serum with c-ANCA from a patient with Wegener's granulomatosis. (c and d) A fine homogeneous rim-like staining of the perinuclear cytoplasm was obtained with a serum containing p-ANCA from a patient with microscopic polyangiitis. (e and f) Atypical p-ANCA ‘type A’ gave a broad inhomogeneous rim-like staining of the nuclear periphery along with scattered intranuclear fluorescent foci (arrows). The atypical p-ANCA ‘type A’ were found in the serum of a patient with primary sclerosing cholangitis. (g and h) In addition to the characteristic staining pattern of atypical p-ANCA ‘type A’, atypical p-ANCA ‘type B’ produced a diffuse cytoplasmic fluorescence. The atypical p-ANCA ‘type B’ were detected in a serum from a patient with autoimmune hepatitis. Note that p-ANCA (c,d) can be distinguished from atypical p-ANCA (e–h) in that the atypical p-ANCA show multiple intranuclear fluorescent foci representing stained invaginations of the multilobulated neutrophil nuclear envelope. Size bars indicate 10 µm.

Fig. 2

Double immunofluorescence labelling of neutrophils by atypical p-ANCA and antibodies against lamin B1 examined by serial horizontal sectioning of human neutrophils using confocal laser scanning microscopy. Atypical p-ANCA were detected with FITC-conjugated goat antihuman IgG secondary antibodies on ethanol-fixed neutrophils. Antibodies against lamin B1 were visualized by Texas red-conjugated goat antirabbit IgG secondary antibodies. When the fluorescence signal of atypical p-ANCA (green) and the fluorescence labelling of antibodies against lamin B1 (red) were optically superimposed, areas of colocalization were indicated by a yellow labelling. Human neutrophils were optically sectioned from the top to the bottom of the cell into horizontal sections of 1 µm using confocal laser scanning microscopy. The left panels (a, b, c, d) show the different staining patterns produced by atypical p-ANCA with respect to the serial horizontal sections of the neutrophil; in the middle panels (f, g, h, i) the fluorescence labelling given by antibodies to lamin B1 is shown; in the right panels (k, l, m, n), the correspondig fluorescence signals produced by atypical p-ANCA and antilamin B1 are optically superimposed. The images of the panels e, j and o represent a three-dimensional reconstruction of the corresponding serial horizontal cell sections. The positions of the horizontal sections with respect to the images shown in the above panels are marked by alphabetic letters as indicated in the corresponding fluorescence images. An additional three dimensional reconstruction of serial vertical sections with particular respect to the intranuclear fluorescence is indicated by A, B and C in the panels e, j and o. The corresponding individual fluorescence images of vertically sectioned neutrophils are not shown. Panels a–e: ethanol-fixed neutrophils were incubated with the serum from a patient with PSC which contained atypical p-ANCA. An inhomogeneous rim-like staining pattern along with intranuclear fluorescent foci given by atypical p-ANCA was detected in each horizontal section of the neutrophils. The three dimensional reconstruction of these images (e) and the three-dimensional reconstruction of vertical sections of the same images clearly assigned the intranuclear labelling to an invagination of the nuclear periphery. Panels f–j: antibodies against lamin B1 gave a rim-like staining of the nuclear envelope along with intranuclear labelling which corresponded to an infolding of the nuclear envelope (j). Panels k–o: the fluorescence signals of atypical p-ANCA and antibodies to lamin B1 completely co-localized in the corresponding serial sections of the neutrophils as well as in the three-dimensional reconstructions. The size bars in the micrographs indicate 10 µm.

Fig. 3

Microscopic immunofluorescence patterns of p-ANCA compared to atypical p-ANCA in relationship to the neutrophil fixation. Antineutrophil antibodies bound to their target proteins were detected by FITC-conjugated goat antihuman IgG secondary antibodies. To visualize the different fluorescence patterns, confocal laser scanning microscopy was used. The schematic diagram shows the subcellular distribution of the target antigen (•) of p-ANCA and atypical p-ANCA with respect to the used fixative agent. (a) On ethanol-fixed neutrophils, p-ANCA gave a fine perinuclear fluorescence labelling with the responsible target proteins collapsing in the perinuclear cytoplasm. (b) Using the cross-linking fixative formaldehyde, p-ANCA diffusely labelled the cytoplasm of ethanol-fixed neutrophils. A highlighting of the fluorescence between the nuclear lobes as frequently seen with c-ANCA on ethanol-fixed neutrophils was not observed. As depicted in the schematic diagram, the target proteins are diffusely distributed throughout the cytoplasm when formaldedhyde is used as fixative. (c) On ethanol-fixed neutrophils, atypical p-ANCA react with neutrophil-specific proteins in the nuclear periphery of neutrophils and produce a broad rim-like fluorescence pattern along with multiple intranuclear fluorescent foci. (d) The localization of the nuclear antigen recognized by atypical p-ANCA is not affected by the formaldehyde fixation. The staining pattern produced by atypical p-ANCA on formaldehyde-fixed neutrophils is identical to that on ethanol-fixed neutrophils. Size bars in the micrographs indicate 10 µm.

Fig. 4

Microscopic immunofluorescence patterns of p-ANCA and atypical p-ANCA on formaldehyde-fixed neutrophils with respect to propidium iodide counterstaining. p-ANCA and atypical p-ANCA were detected using FITC-conjugated goat antihuman IgG secondary (dark grey). Propidium iodide counterstaining, was used to label the nucleus (light grey). When both images were optically superimposed, a co-localization of both fluorescence signals was indicated by a bright white staining. The fluorescence patterns were visualized by confocal laser scanning microscopy. (a) Atypical p-ANCA gave a rim-like labelling of the nuclear periphery along with scattered intranuclear fluorescent foci which both colocalized with the propidium iodide staining. (b) p-ANCA showed a diffuse labelling of the cytoplasm which did not overlap with the nuclear propidium iodide staining. Size bars indicate 10 µm.