Clin. exp. Immunol. (1979) 38, 424-435.

In vivo anti-nuclear antibodies in epithelial biopsies in SLE and other connective tissue diseases J. VIVIAN WELLS, J. WEBB, MARIA VAN DEVENTER, BEVERLEY FRY, K. M. POLLARD, JULIA RAFTOS, WENDY MONK & D. S. NELSON Kolling Institute of Medical Research and Sutton Rheumatism Research Laboratory, Royal North Shore Hospital of Sydney, St Leonards, New South Wales 2065, Aastralia

(Accepted for publication 30 April 1979)

SUMMARY

In vivo anti-nuclear antibody (ANA) was observed by direct immunofluorescence microscopy in epithelial cell nuclei in forty-four biopsies from thirty-three patients. The tissue containing the ANA was macroscopically normal in twenty-seven patients. The thirty-three patients with in vivo biopsy ANA included twenty-three with SLE, three with mixed connective tissue disease, two each with multi-system Sjdgren's syndrome, dermatomyositis, and progressive systemic sclerosis, and one with rheumatoid arthritis. Features of sicca syndrome were noted in seventeen patients. The patterns of the in vivo biopsy ANA in the thirty-three patients were speckled (21), homogeneous (6), nodular (2), and both speckled and homogeneous (4). Complement was not detected in the epithelial cell nuclei. Immunoglobulin(s) and/or complement were deposited along the dermoepidermal junction in thirty-two of the forty-four biopsies, and in dermal blood vessels in twenty-two biopsies. Each patient had serum ANA against rat liver substrate; twenty-seven had high titre ANA (1 in 1000 or greater). Elevated levels of DNA-binding were found in twenty patients (61%), but the level of DNA-binding did not correlate with the intensity of in vivo biopsy ANA staining. Serum antibody to ribonucleoprotein (RNP) was present in eight of the twenty-three patients tested (350 ) all eight patients having clinical features of sicca syndrome. Hypocomplementaemia was found in thirteen patients (400 ) all of whom had active SLE. In vivo biopsy ANA appears to be a real phenomenon of unknown aetiology, and not an artifact, which is found in some patients with active multisystem autoimmune disease, especially SLE.

INTRODUCTION Anti-nuclear antibodies (ANA) can combine with nuclei in tissues and be visualized in biopsy sections treated with fluorescein-labelled anti-immunoglobulin reagents. The phenomenon is also referred to as 'in vivo' ANA. Tissues reported with ANA staining include skin lesions in SLE patients (Tan & Kunkel, 1966; Darley & Munro, 1977), diseased kidneys in SLE (Freedman & Markowitz, 1962; Paronetto & Koffler, 1965; Hench, Tan & Wilson, 1969; McCoy, 1972), pneumocytes and pleural cells in SLE pneumonitis (Pertschuk et al., 1977), and clinically uninvolved skin in autoimmune diseases (Baart de la Faille-Kuyper, 1974; Gilliam, Smiley & Ziff, 1975; Shu et al., 1977; Prystowsky & Tuffanelli, 1978; Prystowsky, Gilliam & Tuffanelli, 1978; Izuno, 1978). The sera of the majority of patients in four of the above studies (Gilliam et al., 1975; Shu et al., 1977; Prystowsky & Tuffanelli, 1978; Izuno, 1978) contained high titres of antibodies to extractable nuclear antigen (ENA). The associations of antibodies Correspondence: Dr J. Vivian Wells, Kolling Institute of Medical Research, Royal North Shore Hospital of Sydney, St Leonards, N.S.W. 2065, Australia. 0009-9104/79/1200-0424S02.00 co 1979 Blackwell Scientific Publications

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to ENA with SLE, mixed connective tissue disease, and other diseases are still under intensive study (Fernandez-Madrid & Mattioli, 1976). The present report describes our clinical, immunological and laboratory observations in thirty-three patients with autoimmune disorders in whom epithelial biopsies contained in vivo ANA. MATERIAL AND METHODS The relevant clinical features of the thirty-three patients are summarized in Table 1. The biopsies were taken fresh by excisional or punch biopsy, frozen, and serial 5 /,m sections cut in AO Cryostat (American Optical Company). Immunofluorescence studies. Antiserum to human IgG was raised in a rabbit, absorbed appropriately, and conjugated with fluorescein isothiocyanate (FITC) by standard methods (Beutner et al., 1970). FITC-conjugated goat antisera to human IgA, IgM and C3 were obtained from Behringwerke AG. The sections were reacted with the above antisera by standard techniques for direct immunofluorescence (Whittingham & Mackay, 1969) and examined with a Nikon fluorescence microscope. The titres and patterns of serum ANA were determined by indirect immunofluorescence with rat liver and human blood film as substrates and an FITC-conjugated polyvalent anti-human immunoglobulin antiserum (Oxford Laboratories). Complement studies. Serum C3 and C4 concentrations were measured by radial immunodiffusion in agar plates containing specific antiserum (Behringwerke AG). Serum haemolytic complement activity (CH50) was measured by a method adapted from Kabat & Mayer (1961) and Kent & Fife (1963). Anti-DNA and anti-RNP antibodies. Antibodies to double stranded deoxyribonucleic acid (DNA) were detected by radioimmunoassay with the Farr Technique and 14C-labelled native DNA (Escherichia coli) provided by the Radiochemical Centre, Amersham (Webb & Whaley, 1974). A normal value with this assay is < 20% binding. Serum antibodies to ribonucleoprotein were detected by counter-immunoelectrophoresis (Bresnihan, Grigor & Hughes, 1977) against a saline-soluble acetone extract of rabbit thymus (Pel-Freeze Biological Inc., USA).

RESULTS The fourty-four biopsies from thirty-three patients with in vivo ANA represented 10% of a total of 427 biopsies from 338 patients with a variety of rheumatological, haematological, dermatological and other diseases. The group included thirty-one females and two males; their ages ranged from 20 to 74 years, with an average of 41 years (Table 1). The thirty-three patients included twenty-three with SLE, three with mixed connective tissue disease, two with dermatomyositis, two with Sj6gren's syndrome of multisystemic type, two with progressive systemic sclerosis and one with rheumatoid arthritis. The twentythree patients with SLE represented 3300 of a total of sixty-nine SLE patients who had skin biopsy performed. The findings on immunofluorescence microscopy in the forty-four biopsies with in vivo ANA are outlined in Table 1. Skin was studied in each case except Case 14 (vagina). Multiple specimens were taken from some patients but only lesional skin was available from six patients. Macroscopically normal tissue was provided from twenty-seven patients. Table 1 also indicates if the site of the biopsy was chronically exposed to sunlight. The nuclear deposits were most clearly seen in epidermal cell nuclei but in some cases dermal cell nuclei were obviously stained. In ten biopsies, in vivo ANA was the only significant finding on immunofluorescence microscopy. The ANA pattern in the biopsy was speckled in twenty-one patients (Figs 1 and 2), homogeneous in six, nodular in two, and both speckled and homogeneous in four (Fig. 3). Complement was not found staining epidermal cell nuclei in any of the biopsies. IgG-class ANA was detected in each of the forty-four biopsies and in fifteen cases it was subjectively graded as heavy in intensity. IgA-class ANA was also found in eight biopsies (one heavy intensity) and IgM-class ANA in fourteen biopsies (three heavy intensity). ANA of all three immunoglobulin classes was found in seven biopsies (four patients with SLE, two with mixed connective tissue disease and one with dermatomyositis). In five of these cases the skin was clinically normal. Significant deposition of immunoglobulins and/or C3 was found at the dermoepidermal junction in thirty-two of the forty-one biopsies. The pattern was granular in each case although it varied greatly in intensity and width. Ten biopsies had significant deposits of both immunoglobulin(s) and C3, 18 had only immunoglobulin(s), and four had only C3. Thus, IgG was deposited at the dermoepidermal

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