Journal of lmmunological Methods, 143 (1991)57-68
57
© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100290A JIM06066
A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis W.J. M a y e t 1, E. H e r m a n n 1, E. C s e r n o k 2, A. K n u t h 1, T. P o r a l l a 1, W.L. G r o s s 2, K.H. M e y e r z u m B i i s c h e n f e l d e 1 1 I. Medizinische Klinik, Universitiit Mainz, F.R.G., 2 Medizinische Universitdt Liibeck und Rheumaklinik, Bad Bramstedt, F.R.G.
(Received 19 February 1991, revised received 15 April 1991, accepted 29 May 1991)
Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (C-ANCA), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in Wegener's granulomatosis (WG). Till now, human neutrophil granulocytes have represented the major antigen source used to detect antibodies in W G by the immunofluorescence technique (IFT). We have tested serum samples of 164 patients with different connective tissue diseases (50 suffering from clinically active WG) performing IFT on a human renal cancer line ( S K - R C l l ) and have found antibodies against the nuclear and cytoplasmic antigens in 39 patients. C-ANCA + sera displayed a characteristic diffuse cytoplasmic staining pattern. Antibody titers measured with human granulocytes were comparable to titers obtained using culture cells. Antibody binding could be inhibited by preabsorption with an extract of human granulocytes or purified proteinase 3. A protein of 29 kDa MW could be isolated by affinity purification using a S K - R C l l extract and a high-titer C-ANCA + serum and antigenic identity was further confirmed by IFT using a monoclonal antibody to proteinase 3. Treatment of tumor cells with cytokines (interferon, tumor necrosis factor) led to a time dependent translocation of the antigen into the nucleus and back to the cytoplasm. The antigen was also expressed on the surface of live cells colocalized with M H C II. In addition, 21 W G patients had antibodies to cytoplasmic organelles identified by laser scanning microscopy as secretory vesicles of the Golgi complex, and five had antibodies to nuclear antigens. This is, to the best of our knowledge, the first report of proteinase 3 in human non-leukemic cells. Our data demonstrate, that the repertoire of antigens recognized by antibodies in WG sera is not
Correspondence to: K.-H. Meyer zum Biischenfelde, I. Medizinische Klinik und Poliklinik der Johannes Gutenberg-Universit~it, Langenbeckstrasse 1, D-6500 Mainz, F.R.G. Abbreviations: WG, Wegener's granulomatosis; ANCA, anti-neutrophilocytecytoplasm antibodies; IFT, indirect immunofluorescence technique; SS, Sj6gren's syndrome; MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; PBC, primary biliary cirrhosis; RF, rheumathoid factor; RA, rheumatoid arthritis; HBD, healthy blood donors; FCS, fetal calf serum; PBS, phosphate-buffered saline; IFN, interferon-y; TNF, tumor necrosis factor; PMSF, phenyl-methyl-sulfonyl-fluoride;ANA, anti-nuclear antibodies; ENA, extractable nuclear antigens; CIE, counterimmunoelectrophoresis;dsDNA, double stranded DNA; AMA, anti-mitochondrial antibodies; SMA, smooth muscle antibodies; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; PAGE, polyacrylamidegel electropboresis; mAb, monoclonal antibody; BSA; bovine serum albumin; PMN, polymorphonuclearleukocytes.
58 limited to human neutrophils and monocytes and indicates a possible functional role of the antigenic proteins. Key words: Wegener's granulomatosis;Anti-neutrophil cytoplasmicantibody; Proteinase 3; Cancer cell line; Antigen shuttling;
Immunofluorescencetechnique
Introduction
Patients and methods
Wegener's granulomatosis (WG) was first described in the 1930s (Wegener 1936) and is now recognized as a distinct type of systemic vasculitis (Fauci et al., 1973; Gross, 1989). Laboratory findings are usually nonspecific indicating a systemic inflammatory illness, but the detection of autoantibodies directed against cytoplasmic antigens of human neutrophil granulocytes and monocytes (anti-cytoplasmic antibodies (ACPA), anti-neutrophilocyte cytoplasm antibodies (ANCA)) in patients with WG have added a new dimension to the diagnostic and pathogenetic aspects of this disease (Van der Woude et al., 1985). Using the indirect immunofluorescence technique (IFT) two different ANCA types have been described. One ANCA type, C-ANCA, produces cytoplasmic staining and is usually specific for the serine proteinase, proteinase 3 (Liidemann et al., 1990). The second ANCA type produces artifactual perinuclear staining on alcohol-fixed neutrophils and has specificity not only for myeloperoxidase (P-ANCA) (FALK et al., 1990), but also for elastase (Goldschmeding et al., 1989; N~issberger et al., 1989) and lactoferrin (Thompson et al., 1989). Whilst searching for an optimized antigen source to detect autoantibodies in patients with connective tissue diseases we investigated a human cancer cell line designated SK-RC 11 growing as a monolayer and easy to keep in long time culture. When screening hundreds of sera we noted that C-ANCA + WG patients exclusively displayed a characteristic diffuse cytoplasmic fluorescence pattern on the renal cancer cell line SK-RCll and, independent of ANCA, nuclear antigens and cytoplasmic organelles were also recognized. In this study the antigenic repertoire of this tumour cell line recognized by WG sera has been investigated further.
Patient's sera
Serum samples were obtained from 300 donors. 50 suffered from clinically active WG. The diagnosis was established on the basis of classical symptoms and the typical histological findings in biopsy specimens as earlier described (N611e et al., 1989). 36 of them were C-ANCA + and no P-ANCA were detected. Eight patients suffered from histologically confirmed Sj6gren's syndrome (SS), three had mixed connective tissue disease (MCTD), 20 had scleroderma, 23 had systemic lupus erythematosus (SLE) according to the revised criteria of the American Rheumatism Association, 11 suffered from primary biliary cirrhosis (PBC) and 49 from rheumatoid factor (RF) positive rheumatoid arthritis (RA). 136 healthy blood donors (HBD) served as controls. All serum samples were stored at - 80° C until used. Cell culture
Human kidney carcinoma cells (SK-RC11) (Ueda et al., 1979) were grown on culture slides (Lab-Tek, Miles Scientific, Naperville, IL) in a humidified CO 2 (5%) atmosphere using DMEM (supplemented with 1% glutamine, 1% penicillin/streptomycin (Serva, Heidelberg, F.R.G.), 1% sodium-pyruvate (Seromed, Berlin, F.R.G.)) and 10% fetal calf serum (FCS) (Biochrom, Berlin, F.R.G). Cells were used after three passages in cell culture. The cells were washed with 0.15 M phosphate-buffered saline pH 7.4 (PBS) and fixed with ethanol 96%, methanol (abs) or paraformaldehyde 3.7% (at - 2 0 ° C for 1 h). To test the influence of different cytokines on antigen expression, 100 I U / m l interferon-7 (IFN) (Sigma, Deisenhofen, F.R.G.) and 1 ~ g / m l tumor necrosis factor (TNF) (Boehringer Mannheim, F.R.G.) were added to the medium prior to fixation. Cells were incubated for different inter-
59
suits documented by photography with Ilford XP-1 film. Antibodies to extractable nuclear antigens (ENA) were detected using Ouchterlony immunodiffusion and counterimmunoelectrophoresis (CIE) (Tan et al., 1982). Sera were screened by Ouchterlony immunodiffusion of undiluted sera with an antigen concentration of 50 mg/ml in 0.65% agarose gel (Nilsson, 1983). Rabbit thymus extract (Paesel, Frankfurt, F.R.G.) and human spleen extract prepared as described by Venables et al. (1983) served as antigens. CIE was performed as described by Tan (Tan et al., 1982) using standardized reference antisera (anti-Ro, anti-La, anti-Sm, anti-RNP, Centers for Disease Control, Atlanta, CA). Antibodies to doublestranded DNA (dsDNA) were determined by Farr and PEG assays (Smeenk et al., 1982) and by 1FT on Crithidia luciliae (Antibodies, Davis, CA). Anti-mitochondrial antibodies (AMA) and smooth muscle antibodies (SMA) were detected using IFT on rat tissue. C-ANCA were detected according to the method of Van der Woude et al. (1985) and by enzyme-linked immunosorbent assay (ELISA) (a extract) as described by Rasmussen et al. (1990). Rheumatoid factor (RF) was determined by a latex fixation test (Behringwerke, Marburg, F.R.G.). IgG was prepared from C-ANCA + sera by ammonium sulfate precipitation and ion exchange chromatography on DEAE-Sephadex (Pharmacia, Uppsala, Sweden). To minimize nonspecific fluorescent staining and
vals ranging from 5 to 300 min and fixed thereafter with ethanol.
Preparation of SK-RCll extracts SK-RCll cells were grown as described above in cell culture flasks at a cell density of 4 x 104 cells/cm 2. 108 cells were harvested after trypsin treatment (0.5 m g / m l for 5 min) by centrifugation at 800 x g for 5 min, washed with PBS and resuspended in 2 ml PBS containing 1 mM protease inhibitor phenyl-methyl-sulfonyl-fluoride (PMSF). Cell extracts were obtained by homogenization of the cells after freezing and thawing using a Dounce Potter homogenizer with an Spestle (30 strokes). Cell debris was removed by centrifugation (10,000 X g for 30 min at 4° C) and the supernatant used as a total extract. Antibody testing Antinuclear antibodies (ANA) were detected using IFT on rat tissue and HEp2 cells. HEp2 cells were commercially prepared (Antibodies, Davis, CA) and stained tissues were analyzed with a Zeiss Axiophot microscope equipped with epifluorescence optics (Zeiss, Oberkochen, F.R.G.). A super-pressure mercury lamp (HBO 50) served as a light source. Control experiments revealed that an excitation filter BP 485/20 and a cut-off filter LP 520 were specific for the fluorochrome dye used (data not shown). In addition, a Zeiss LSM 10 laser scan microscope (Zeiss, Oberkochen, F.R.G.) was employed and the reTABLE I ANTIBODIES DETECTED WITH DIFFERENT METHODS Diagnosis
Number of patients
Antibodies detected
IFT rat tissue
IFT HEp
IFT SK-RC11
Crit lucil.
CIE
Farr PEG
Neutr. Gran.
ANCA ELISA
WG SLE Scleroderma SS PBC MCTD RA(RF +) HBD
50 23 20 8 11 3 49 136
ANCA, Gol, ANA dsDNA, La Nucl, Ro, Gol Ro/La AMA, SMA ANA -
0 23 5 2 9 2 0 0
3 23 16 7 11 3 0 0
39 23 20 8 11 3 4 0
0 23 0 0 0 0 0 0
0 5(La) 4(Ro) 8 0 0 0 0
0 23 0 0 0 0 0 0
36 0 0 0 0 0 0 0
34 0 0 0 0 0 0 0
Key: WG, Wegener's granulomatosis; SLE, systemic lupus erythematosus; SS, Sj6gren's syndrome; PBC, primary biliary cirrhosis; MCTD, mixed connective tissue disease; RA, rheumatoid arthritis; HBD, healthy blood donors; ANA, anti-nuclear antibodies; dsDNA, antibodies to double stranded DNA; Nucl, antibodies to nucleoli; Gol, antibodies to the Golgi region; AMA, antimitochondrial antibodies; SMA, smooth muscle antibodies.
60
A
29
13
C
w,
i
Fig. 2. PAGE and Western blot. A: affinity purification usm a SK-RCll extract and a high titre C-ANCA + serum of WG patient; B: reaction of two C - A N C A + sera in a Western blot (29 kDa MW); C: reaction of a healthy blood donor (control) in a Western blot.
to e x c l u d e Fc r e c e p t o r b i n d i n g , ( F a b ' ) 2 f r a g m e n t s w e r e p r e p a r e d as d e s c r i b e d b y G a r v e y e t al. (1977). F i x e d S K - R C l l c e l l s w e r e i n c u b a t e d w i t h patients' sera or purified IgG fractions diluted
Fig. 1. IFT with a monospecific C-ANCA ÷ serum of a patient with WG (titre 1/640) (original magnification 630×). A: reaction on human neutrophils; B: reaction on SK-RCll cells; C: weak reaction on SK-RCll cells after preabsorption of antibodies with purified proteinase 3; D: weak reaction on human neutrophil granulocytes after preabsorption of antibodies with an extract of SK-RCll cells.
61 1 / 4 0 in PBS for 1 h in a humid c h a m b e r at room t e m p e r a t u r e . A f t e r extensive washing with PBS the cells were incubated with the second antibody, fluorescein isothiocyanate ( F I T C ) - c o n j u g a t e d anti-human I g G (F-5512, Sigma, Deisenhofen). Sections were then washed again and
incubated with Mowiol (Mowiol 4-88, H6chst, F r a n k f u r t F.R.G.). Live cells were stained by adding 10 #1 of undiluted serum or purified l g G fraction to the medium. Cells were washed with PBS and fixed immediately. M H C II expression was tested by incubation with a monoclonal anti-
Fig. 3. IFT on SK-RCll tumor cells (original magnification 630 x). A: reaction of a monospecificC-ANCA+ serum of a patient with WG (titre 1/640); B: reaction of 29 kDa antibody eluted from nitrocellulose (see Fig. 2); C: reaction of a monoclonal antibody to proteinase 3 (WGM2).
62 D R antibody (Becton Dickinson, Mountain View, CA). Affinity purified C - A N C A antigen was prepared as previously described (Liidemann et al., 1988) using an extract of human neutrophils or S K - R C l l cells. Proteinase 3 was purified as described by Kao et al. (1988). Polyacrylamide gel electrophoresis ( P A G E ) and Western blotting were performed as described previously (Bachmann et al., 1986; Mayet et al., 1988). For inhibition experiments serum of a C - A N C A + patient (1:640) was mixed v / v with affinity purified CA N C A antigen diluted to 0.1 m g / m l protein concentration in PBS or purified proteinase 3 and incubated on a rotator for 1 h at 3 7 ° C and overnight at 4 o C. The mixture was centrifuged at 30,000 × g for 15 min at 4 ° C and the supernatants kept as absorbed sera. A murine monoclonal antibody (mAb) to proteinase 3 designated W G M 2 (Csernok et al., 1990) was used as a control in the IFT.
Elution of antibodies from nitrocellulose Antibodies were eluted from nitrocellulose as described by McHugh et al. (1990) with minor modifications. Briefly, a probed strip of nitrocellulose with the band at 29 kDa was lined up accurately against an untreated native strip. A band was cut from this native strip corresponding to the area of reactivity on the stained strip. A band corresponding to an unstained area on the probed strip was also cut from the native sheet to act as a negative control. The bands were cut into 3 mm 2 pieces and placed into separate Eppendorf tubes. Each tube was filled with P B S / 0 . 0 5 % Tween (pH 7.3) and gently agitated for 10 min. This was followed by two washes with PBS and a
Fig. 4. Time-dependent antigen translocation after treatment of SK-RCll tumor cells with cytokines (IFN, TNF). IFT on fixed cells (original magnification 630 x ). A: weak cytoplasmic fluorescence and perinuclear concentration after 30 min of cytokine treatment; B: perinuclear concentration and weak nuclear fluorescence after 60 min of cytokine treatment; C: bright nuclear fluorescence after 75 rain of cytokine treatment; D: cytoplasmic redistribution after 135 min of cytokine treatment; E: reaction with monoclonal antibody to proteinase 3 (WGM2) after 120 min of cytokine treatment; F: incubation with a monospecific C-ANCA + serum after preabsorption of antibodies with purified proteinase 3: no reaction.
63
Fig. 5. Time-dependent antigen translocation after treatment with cytokines (IFN, TNF). IFT on fixed (A, B, D) and live (C) SK-RCll2 tumor cells; laser scanning microscopy; n = nucleus (original magnification 1000 x). A: weak nuclear fluorescence and perinuclear concentration; B: nuclear fluorescence; C: membrane expression; D: reaction with secretory vesicles leaving the trans-region of the Golgi complex.
wash with 0.25 M KC1 in PBS. After the last wash, 0.5 ml of the elution buffer (0.2 M glycine, p H 2.8) containing 1 m g / m l bovine serum albu-
min (BSA) was added to each tube and the contents agitated for 2 min. 1 M Tris was added to bring the p H back to neutral, the nitrocellulose
64
pieces were stored for further use and the eluates transferred to dialysis tubing for dialysis against large volumes of cold PBS.
Results
Antibodies detected by IFT on SK-RC11 cells recognized different nuclear and cytoplasmic antigens, as shown in Tables I and II. Staining patterns of antibodies of patients with LE, SS, scleroderma, MCTD and PBC were similar to the patterns obtained using rat tissue or HEp2 cells. Compared to standard procedures, sensitivity with S K - R C l l cells was slightly increased, especially for ANA (see also Table I). The most striking and unexpected result was that C-ANCA + sera of W G patients displayed a unique and characteristic diffuse cytoplasmic staining pattern (Fig. 1B). Using cultured cells antibody titres were comparable to the titres obtained with human granulocytes. There was no difference between ANCA + serum or a purified IgG fraction and modification of the fixation procedure (time, methanol, ethanol or paraformaldehyde) did not alter the binding pattern. C-ANCA + sera were almost negative on SKR C l l cells or human neutrophils when preabsorbed by incubation with an extract of human neutrophil granulocytes, S K - R C l l cells or purified proteinase 3 (Figs. 1C and 1D). A well
TABLE II ANTIBODIES D E T E C T E D BY IFT ON S K - R C l l T U M O R CELLS Diagnosis
WG
Number of patients 50
SLE 23 Sclerod. 20 SS 8 PBC 11 MCTD 3 R A (RF + ) 49 HBD 136
IFT-pattern on S K - R C l l cells
36 diff. cytoplasm, 21 Golgi, 5 ANA (2 homo, 3 speckled) 23 A N A (18 homo, 5 speckled) 20 A N A (20 nucleolar) 8 A N A (speckled) 11 AMA, 3 SMA 3 A N A (1 speckled, 2 centromer) 4 SMA 0
Fig. 6. IFT on live unfixed S K - R C l l cells incubated with purified IgG fraction of a C-ANCA + patient (original magnification 630 ×). Membrane expression of the antigen (arrow).
characterized mAb to proteinase 3 (WGM2) also produced the diffuse cytoplasmic staining pattern (Fig. 3C). Following affinity purification using a SK-RC11 extract and a high-titre C-ANCA + serum from a WG patient a protein of 29 kDa MW was isolated (Fig. 2A). This protein was recognized by C-ANCA + W G sera in a Western blot (Fig. 2B). Bound antibodies could be eluted from the nitrocellulose and again revealed the typical diffuse cytoplasmic staining pattern when performing IFT on S K - R C l l (Fig. 3B). Even after several passages in cell culture no changes in antigen expression could be observed. Treatment of live cells during culture with IFN and T N F led to a time-dependent translocation of the antigen (Figs. 4 and 5). After 30-45 min the antigen was concentrated in the perinuclear region and 30 min later it was found in the nucleus. The redistribution of antigen into the cytoplasm started 60 min later. After 120-240 min the antigen was detected on the surface of live cells (Fig. 6) colocalized with MHC II (DR) (data not shown). Similar staining patterns were seen after incubation with C-ANCA + sera or WGM2 (Fig. 4E). In addition, 21 of the 50 WG sera and five of the 20 scleroderma sera were shown to possess antibodies to cytoplasmic or-
65 ganelles, presumably secretory vesicles leaving the trans-region of the Golgi complex (Fig. 5D).
Discussion
In the early 80s, several groups independently reported the occurrence of antibodies directed against cytoplasmic antigens of neutrophils (ANCA) in patients with vasculitis and glomerulonephritis (Davies et al., 9182; Hall et al., 1984; Van der Woude et al., 1985). Subsequently, ANCA have proved to be a useful clinical tool in supporting the diagnosis and monitoring disease activity in WG (N611e et al., 1989; Cohen-Tervaert et al., 1990). Human neutrophil granulocytes and the promyelocytic HL-60 cell line have represented the exclusive sources of antigen for detecting autoantibodies in WG sera since studies have failed to reveal proteinase 3 in cells other than neutrophils and monocytes (Braun et al., 1991). Crude extracts of granulocytes have been used to characterize the relevant antigens by affinity chromatography with high titre ANCA + sera or monoclonal antibodies (Goldschmeding et al., 1988, 1990; Daha et al., 1990; Liidemann et al., 1990). Moreover, ANCA may play a role in pathogenesis by releasing proteolytic enzymes and O 2 radicals from neutrophil granulocytes (Falk et al., 1990). In this study we have tested 50 sera of patients with WG by performing IFT on a human renal cancer line SK-RCll and have found antibodies against nuclear and cytoplasmic antigens. In 39 patients C-ANCA + sera reacted with a characteristic diffuse cytoplasmic pattern on tumor cells and antibody binding could be inhibited by preabsorption with purified proteinase 3. Treatment of cells with cytokines led to a translocation of the antigen and this was further confirmed by cross-absorption experiments to exclude reactions with a different up-regulated nuclear antigen. The mechanism of this time-dependent translocation of the C-ANCA antigen triggered by cytokines such as IFN remains unclear. A similar antigen shuttling has recently been shown for the La/SS-B antigen in CV-1 cells (Bachmann et al., 1989). The antigen may be cotrans-
ported together with other autoantigens and membrane expression of the antigen could mediate the recognition of a cytoplasmic antigen by the host thereby enabling circulating autoantibodies to bind to these cells in vivo. Recently, we have demonstrated by immunoelectron microscopy the presence of C-ANCA antigen proteinase 3 in small amounts on the plasma membrane of resting normal human neutrophils and monocytes (Csernok et al., 1990). In contrast to the present results, we did not see membrane staining by IFT on unstimulated SK-RCll cells. However, it should be pointed out that immunoelectron microscopy is more sensitive than IFT and this may explain the different findings. Our preliminary data do at least hint at a possible functional role of the antigenic protein. Inhibition experiments with a crude extract of granulocytes, SK-RCll cells and purified proteinase 3 showed identity between the antigen recognized in neutrophils and SK-RCll. Antigenic identity was further confirmed by IFT using a mAb to proteinase 3. Proteinase 3 was described by Baggiolini et al. in 1978 as a result of observations made after nondenaturing PAGE of an extract of azurophilic granules from polymorphonuclear leucocytes (PMN) (Baggiolini et al., 1987). It is a neutral serine proteinase and has recently been shown to be important in the pathogenesis of human emphysema (Kao et al., 1988). Proteinase 3 is identical with myeloblastin, a serine protease whose complementary DNA has been cloned from promyelocytic leukaemia HL-60 cells. It appears to possess multiple biological activities, microbicidal and elastinolytic functions and a regulatory function during myelomonocytic cell differentiation (Jenne et al., 1990). This is, to our knowledge, the first report of the expression of this serine proteinase in nonleukemic human tumor cells. The antigen repertoire in SK-RC11 tumor cells seems to overlap with that of human neutrophils (Fig. 7). Despite the apparent differences between active tumor cells and neutrophils there are several common functional aspects. In order to pass through the blood vessel walls neutrophils adhere to the endothelial cells, squeeze between the ceils and through the basement membrane. The azurophilic granules of neutrophils are equivalent
66
Fig. 7. Comparison of IFT pattern on human neutrophil granulocytes and on SK-RC11 tumor cells (original magnification 630 x ). A: C-ANCA + serum of a WG patient on human granulocytes; B: C-ANCA + serum as used in A on S K - R C l l tumor cells (note characteristic diffuse cytoplasmic fluorescence and additional reaction with Golgi organelles); C: C-ANCA serum of a WG patient on human granulocytes; D: C - A N C A - serum as used in C on S K - R C l l tumor cells (note bright nucleolar fluorescence despite negative reaction on human granulocytes).
67 to t h e l y s o s o m e s in o t h e r cell types, i.e., t h e y c o n t a i n acid h y d r o l a s e s . O b v i o u s l y t h e s e e n z y m e s c a n cleave a large n u m b e r o f d i f f e r e n t tissue c o n s t i t u e n t s which a r e i m p o r t a n t for t h e struct u r e of t h e e x t r a c e l l u l a r m a t r i x o f c o n n e c t i v e tissue a n d for the s h a p e , m i g r a t i o n , g r o w t h a n d d i f f e r e n t i a t i o n o f cells ( R o o s et al., 1990). T h e invasion o f n o r m a l tissues a n d t h e p e n e t r a t i o n of v a s c u l a r b a s e m e n t m e m b r a n e s by m e t a s t a t i c tum o r cells m a y also r e q u i r e t h e active p a r t i c i p a tion o f hydrolytic enzymes. L y s o s o m a l c a t h e p s i n s have b e e n r e p o r t e d l y e l e v a t e d in s o m e t u m o r tissues a n d e n h a n c e d p r o d u c t i o n a n d s e c r e t i o n o f s e r i n e p r o t e a s e s have b e e n a s s o c i a t e d with t h e n e o p l a s t i c t r a n s f o r m a t i o n o f a v a r i e t y of cell t y p e s ( F i d l e r et al., 1989). T h e r e is clearly m u c h m o r e to b e l e a r n e d a b o u t t h e physical a n d f u n c t i o n a l p r o p e r t i e s o f p r o t e i n a s e 3. T h e s e investigations d e m o n s t r a t e t h a t the r e p e r t o i r e o f a n t i g e n s r e c o g n i z e d by a n t i b o d i e s in W G s e r a is not l i m i t e d to h u m a n n e u t r o p h i l s a n d m o n o c y t e s . This fact a g a i n p o i n t s to an a u t o i m m u n e p r o n e c o n d i t i o n in p a t i e n t s with W G (Shilitoe et al., 1974; G r o s s , 1989) a n d suggests a closer r e l a t i o n s h i p b e t w e e n W G a n d o t h e r connective tissue diseases. F u r t h e r i n v e s t i g a t i o n s will b e n e c e s s a r y to evaluate the production and secretion of prot e i n a s e 3 by t u m o r cells a n d to assess t h e diagnostic v a l u e o f I F T on d i f f e r e n t t u m o r cell lines in W G . I n a d d i t i o n , t h e d i f f e r e n t n u c l e a r antigens a n d c y t o p l a s m i c s t r u c t u r e s r e c o g n i z e d by W G s e r a a n d t h e i r m e m b r a n e e x p r e s s i o n n e e d to be c h a r a c t e r i z e d in m o r e detail.
Acknowledgements W e a r e g r a t e f u l for t h e t e c h n i c a l a s s i s t a n c e f r o m Mrs. A. Canisius, Ms. S. Mayer, Ms. J. K a r b a c h a n d Mrs. Sayah. W e wish to t h a n k Professor Dr. M. B a c h m a n n , I n s t i t u t fiir Physiologische C h e m i e , Universit~it M a i n z , for h e l p f u l discussions a n d e x p e r t t e c h n i c a l advice c o n c e r n i n g laser scanning microscopy. We thank Professor Dr. H. L e h m a n n , E v a n g e l i s c h e s K r a n k e n h a u s Z w e i b r i i c k e n , 6660 Z w e i b r i i c k e n , F . R . G . , for p e r mission to r e p o r t p a t i e n t s u n d e r his care.
References Bachmann, M., Mayet, W.J., Schr6der, H.C., Pfeifer, K., Meyer zum Biischenfelde, K.H. and Miiller, W.E.G. (1986) Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells. Proc. Natl. Acad. Sci. U.S.A. 83, 7770. Bachmann, M., Pfeifer, K., Schr6der, H.C. and MiJller, W.E.G. (1989) The La antigen shuttles between the nucleus and the cytoplasm in CV-1 cells. Mol. Cell. Biochem. 85, 103. Baggiolini, M., Bretz, U., Dewald, B. and Feigenson, M.E. (1978) The polymorphonuclear leukocyte. Agents Actions 8,3. Braun, M.G., Csernok, E., Gross, W.L. and Miiller-Hermelink, H.K. Proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener's granulomatosis: immunolocalization in normal and pathological tissues. Am. J. Pathol., submitted. Churg, A. (1983) Pulmonary angiitis and granulomatosis revisited. Hum. Pathol. 14, 868. Cohen Tervaert, J.W., Huitema, R.J. and Sluiter, W.L. (1991) Relapses of Wegener's granulomatosis: prevention by treatment based on antineutrophil cytoplasmic antibody levels. Lancet, in press. Csernok, E., Lfidemann, J., Gross, W.L. and Bainton, D.F. (1990) Ultrastructural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener's granulomatosis. Am. J. Pathol. 137, 1113. Daha, M.R., Leusen, J., Kramps, J.A., Schrama, E., Van Es, L.A. and Van der Woude, F.J. (1990) Isolation from purulent sputum of an antigen reactive with antibodies in serum of patients with Wegener's granulomatosis. Neth. J. Med. 36, 117. Davies, D.J., Moran, J.E., Niall, J.F. and Ryan, G.B. (1982) Segmental necrotizing glomerulonephritis with antineutrophil antibody: possible arbovirus aetiology? Br. Med. J. 285, 606. Falk, R.J., Terrell, R.S., Charles, L. and Jennette, J.C. (1990) Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc. Natl. Acad. Sci. U.S.A. 87, 4115. Fauci, A.S. and Wolff, S.M. (1973) Wegener's granulomatosis: studies in eighteen patients and a review of the literature. Medicine (Baltimore) 52, 535. Fidler, I.J. and Hart, I.R. (1989) Principles of cancer biology: cancer metastatis. In: V.T. De Vita (Ed.), Cancer-Principles and Practice of Oncology, 2nd edn. Lippincott., Philadelphia, PA. Garvey, J.S., Cremer, N.C. and Sussdorf, D.H. (1977) Methods in Immunology. Benjamin, Reading, MA. Goldschmeding, R., Van der Schoot, C.E., Cohen Tervaert, J.W., Mason, D.Y., Van dem Borne, A.E.G.K. and Kallenberg, C.G.M. (1988) Autoantibodies against myeloid lysosomal enzymes: a novel class of autoantibodies associated with vasculitic syndromes. Kidney Int. 34, 558. Goldschmeding, R., Cohen-Tervaert, J.W. and Van der Schoot, C.E. (1989) ANCA, anti-myeloperoxidase and anti-elastase: three members of a novel class of autoantibodies against myeloid lysosomal enzymes. APMIS 97, 48.
68 Goldschmeding, R., Cohen Tervaert, J.W., Gans, R.O.B., Dolman, K.M., Van den Ende, M.E., Kuizinga, M.C., Kallenberg, C.G.M. and Van dem Borne, A.E.D.G.K. (1990) Different immunological specifities and disease associations of C-ANCA and P-ANCA. Neth. J. Med. 36, 114. Gross, W.L. (1989) Wegener's granulomatosis: the aspects of disease course, immunodiagnostic procedures and stageadapted treatment. Sarcoidosis 6, 15. Hall, J.B., Waldham, B.M., Wood, C.J., Ashton, V. and Adam, W.R. (1984) Vasculitis and glomerulonephritis: a subgroup with an antineutrophil cytoplasmic antibody. Aust. NZ. J. Med. 14, 277. Jenne, D.E. and Tschopp, J. (1990) Wegener's autoantigen decoded. Nature 346, 520. Kao, R.C., Wehner, N.G., Skubitz, K.M., Gray, B.H. and Hoidal, J.R. (1988) Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J. Clin. Invest. 82, 1963. Liidemann, J., Utecht, B. and Gross, W.L. (1988) Detection and quantitation of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by ELISA using affinitypurified antigen. J. Immunol. Methods 114, 167. Liidemann, J., Csernok, E., Ulmer, M., Lemke, H., Utecht, B., Rautmann, A. and Gross, W.L. (1990a) Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis: immunodiagnostic value, monoclonal antibodies and characterization of the target antigen. Neth. J. Med. 36, 157. Liidemann, J., Utecht, B. and Gross, W.L. (1990b) Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme. J. Exp. Med. 171, 357. Mayet, W.J., Bachmann, M., Pfeifer, K., Schr6der, H.C., Miiller, W.E.G., Gudat, W., Korting, G.W. and Meyer zum Biischenfelde, K.H. (1988) A monoclonal Ro-antibody and the serum of a Ro-positive patient with subacute cutaneous lupus erythematosus (SCLE) react with basal layers of human epidermis. Eur. J. Clin. Invest. 18, 465. McHugh, N.J., James, I.E., Fairburn, K. and Maddison, P.J. (1990) Autoantibodies to mitochondrial and centromere antigens in primary biliary cirrhosis and systemic sclerosis. Clin. Exp: Immunol. 81,244.
N~issberger, L., Johnsson, H. and Sj6hm, A.G. (1989) Circulating anti-elastase antibodies in systemic lupus erythematosus. Lancet i, 509. Nilsson, L.A. (1983) Double-diffusion in gel. Scand. J. Immun. 17, 57. N611e, B., Specks, U., Liidemann, J., Rohrbach, M.S., De Remee, R.A. and Gross, W.L. (1989) Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener's granulomatosis. Ann. Intern. Med. 111, 28. Rasmussen, N., Sj61in, C., Isaksson, B., Bygren, P. and Wieslander, J. (1990) An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA). J. Immunol. Methods 127, 139. Roos, D. and Dolman, K.M. (1990) Neutrophil involvement in inflammatory tissue damage. Neth. J. Med. 36, 89. Shilitoe, E.J., Lehner, T., Lessor, M.H. and Harrison, D.F.N. (1974) Immunological features of Wegener's granulomatosis. Lancet i. 281. Smeenk, R., Van der Lelij, G. and Aarden, L. (1982) Avidity of antibodies to dsDNA: comparison of IFT on Crithidia luciliae, FARR-assay and PEG-assay. J. Immunol. 128, 73. Tan, E.M. (1982) Special antibodies for the study of systemic lupus erythematosus and analysis. Arthritis Rheum. 25, 753. Thompson, R.A. and Lee, S.S. (1989) Antineutrophil cytoplasmic antibodies. Lancet i, 670. Ueda, R., Shiku, H., Pfreundschuh, M., Takahashi, T., Li, L.T.C., Whitmore, W.F., Oettgen, H.F. and Old, L.J. (1979) Cell surface antigens of human renal cancer defined by autologous typing. J. Exp. Med. 150, 564. Van der Woude, F.J., Rasmussen, N., Lobatto, S., Wiik, A., Permin, H., Van Es, L.A., Van der Giessen, M., Van der Hem, G.K. and The, T.H. (1985) Autoantibodies against neutrophils and monocytes. Tool for diagnosis and marker of disease activity in WG. Lancet i, 425. Venables, P., Smith, P. and Maini, R. (1983) Purification and characterization of the Sj6gren's syndrome A and B antigens. Clin. Exp. Immunol. 54, 731. Wegener, F. (1936) Ober generalisierte, septische Gef~isserkrankungen. Verh. Dtsch. Ges. Pathol. 29, 202.
57
© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100290A JIM06066
A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis W.J. M a y e t 1, E. H e r m a n n 1, E. C s e r n o k 2, A. K n u t h 1, T. P o r a l l a 1, W.L. G r o s s 2, K.H. M e y e r z u m B i i s c h e n f e l d e 1 1 I. Medizinische Klinik, Universitiit Mainz, F.R.G., 2 Medizinische Universitdt Liibeck und Rheumaklinik, Bad Bramstedt, F.R.G.
(Received 19 February 1991, revised received 15 April 1991, accepted 29 May 1991)
Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (C-ANCA), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in Wegener's granulomatosis (WG). Till now, human neutrophil granulocytes have represented the major antigen source used to detect antibodies in W G by the immunofluorescence technique (IFT). We have tested serum samples of 164 patients with different connective tissue diseases (50 suffering from clinically active WG) performing IFT on a human renal cancer line ( S K - R C l l ) and have found antibodies against the nuclear and cytoplasmic antigens in 39 patients. C-ANCA + sera displayed a characteristic diffuse cytoplasmic staining pattern. Antibody titers measured with human granulocytes were comparable to titers obtained using culture cells. Antibody binding could be inhibited by preabsorption with an extract of human granulocytes or purified proteinase 3. A protein of 29 kDa MW could be isolated by affinity purification using a S K - R C l l extract and a high-titer C-ANCA + serum and antigenic identity was further confirmed by IFT using a monoclonal antibody to proteinase 3. Treatment of tumor cells with cytokines (interferon, tumor necrosis factor) led to a time dependent translocation of the antigen into the nucleus and back to the cytoplasm. The antigen was also expressed on the surface of live cells colocalized with M H C II. In addition, 21 W G patients had antibodies to cytoplasmic organelles identified by laser scanning microscopy as secretory vesicles of the Golgi complex, and five had antibodies to nuclear antigens. This is, to the best of our knowledge, the first report of proteinase 3 in human non-leukemic cells. Our data demonstrate, that the repertoire of antigens recognized by antibodies in WG sera is not
Correspondence to: K.-H. Meyer zum Biischenfelde, I. Medizinische Klinik und Poliklinik der Johannes Gutenberg-Universit~it, Langenbeckstrasse 1, D-6500 Mainz, F.R.G. Abbreviations: WG, Wegener's granulomatosis; ANCA, anti-neutrophilocytecytoplasm antibodies; IFT, indirect immunofluorescence technique; SS, Sj6gren's syndrome; MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; PBC, primary biliary cirrhosis; RF, rheumathoid factor; RA, rheumatoid arthritis; HBD, healthy blood donors; FCS, fetal calf serum; PBS, phosphate-buffered saline; IFN, interferon-y; TNF, tumor necrosis factor; PMSF, phenyl-methyl-sulfonyl-fluoride;ANA, anti-nuclear antibodies; ENA, extractable nuclear antigens; CIE, counterimmunoelectrophoresis;dsDNA, double stranded DNA; AMA, anti-mitochondrial antibodies; SMA, smooth muscle antibodies; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; PAGE, polyacrylamidegel electropboresis; mAb, monoclonal antibody; BSA; bovine serum albumin; PMN, polymorphonuclearleukocytes.
58 limited to human neutrophils and monocytes and indicates a possible functional role of the antigenic proteins. Key words: Wegener's granulomatosis;Anti-neutrophil cytoplasmicantibody; Proteinase 3; Cancer cell line; Antigen shuttling;
Immunofluorescencetechnique
Introduction
Patients and methods
Wegener's granulomatosis (WG) was first described in the 1930s (Wegener 1936) and is now recognized as a distinct type of systemic vasculitis (Fauci et al., 1973; Gross, 1989). Laboratory findings are usually nonspecific indicating a systemic inflammatory illness, but the detection of autoantibodies directed against cytoplasmic antigens of human neutrophil granulocytes and monocytes (anti-cytoplasmic antibodies (ACPA), anti-neutrophilocyte cytoplasm antibodies (ANCA)) in patients with WG have added a new dimension to the diagnostic and pathogenetic aspects of this disease (Van der Woude et al., 1985). Using the indirect immunofluorescence technique (IFT) two different ANCA types have been described. One ANCA type, C-ANCA, produces cytoplasmic staining and is usually specific for the serine proteinase, proteinase 3 (Liidemann et al., 1990). The second ANCA type produces artifactual perinuclear staining on alcohol-fixed neutrophils and has specificity not only for myeloperoxidase (P-ANCA) (FALK et al., 1990), but also for elastase (Goldschmeding et al., 1989; N~issberger et al., 1989) and lactoferrin (Thompson et al., 1989). Whilst searching for an optimized antigen source to detect autoantibodies in patients with connective tissue diseases we investigated a human cancer cell line designated SK-RC 11 growing as a monolayer and easy to keep in long time culture. When screening hundreds of sera we noted that C-ANCA + WG patients exclusively displayed a characteristic diffuse cytoplasmic fluorescence pattern on the renal cancer cell line SK-RCll and, independent of ANCA, nuclear antigens and cytoplasmic organelles were also recognized. In this study the antigenic repertoire of this tumour cell line recognized by WG sera has been investigated further.
Patient's sera
Serum samples were obtained from 300 donors. 50 suffered from clinically active WG. The diagnosis was established on the basis of classical symptoms and the typical histological findings in biopsy specimens as earlier described (N611e et al., 1989). 36 of them were C-ANCA + and no P-ANCA were detected. Eight patients suffered from histologically confirmed Sj6gren's syndrome (SS), three had mixed connective tissue disease (MCTD), 20 had scleroderma, 23 had systemic lupus erythematosus (SLE) according to the revised criteria of the American Rheumatism Association, 11 suffered from primary biliary cirrhosis (PBC) and 49 from rheumatoid factor (RF) positive rheumatoid arthritis (RA). 136 healthy blood donors (HBD) served as controls. All serum samples were stored at - 80° C until used. Cell culture
Human kidney carcinoma cells (SK-RC11) (Ueda et al., 1979) were grown on culture slides (Lab-Tek, Miles Scientific, Naperville, IL) in a humidified CO 2 (5%) atmosphere using DMEM (supplemented with 1% glutamine, 1% penicillin/streptomycin (Serva, Heidelberg, F.R.G.), 1% sodium-pyruvate (Seromed, Berlin, F.R.G.)) and 10% fetal calf serum (FCS) (Biochrom, Berlin, F.R.G). Cells were used after three passages in cell culture. The cells were washed with 0.15 M phosphate-buffered saline pH 7.4 (PBS) and fixed with ethanol 96%, methanol (abs) or paraformaldehyde 3.7% (at - 2 0 ° C for 1 h). To test the influence of different cytokines on antigen expression, 100 I U / m l interferon-7 (IFN) (Sigma, Deisenhofen, F.R.G.) and 1 ~ g / m l tumor necrosis factor (TNF) (Boehringer Mannheim, F.R.G.) were added to the medium prior to fixation. Cells were incubated for different inter-
59
suits documented by photography with Ilford XP-1 film. Antibodies to extractable nuclear antigens (ENA) were detected using Ouchterlony immunodiffusion and counterimmunoelectrophoresis (CIE) (Tan et al., 1982). Sera were screened by Ouchterlony immunodiffusion of undiluted sera with an antigen concentration of 50 mg/ml in 0.65% agarose gel (Nilsson, 1983). Rabbit thymus extract (Paesel, Frankfurt, F.R.G.) and human spleen extract prepared as described by Venables et al. (1983) served as antigens. CIE was performed as described by Tan (Tan et al., 1982) using standardized reference antisera (anti-Ro, anti-La, anti-Sm, anti-RNP, Centers for Disease Control, Atlanta, CA). Antibodies to doublestranded DNA (dsDNA) were determined by Farr and PEG assays (Smeenk et al., 1982) and by 1FT on Crithidia luciliae (Antibodies, Davis, CA). Anti-mitochondrial antibodies (AMA) and smooth muscle antibodies (SMA) were detected using IFT on rat tissue. C-ANCA were detected according to the method of Van der Woude et al. (1985) and by enzyme-linked immunosorbent assay (ELISA) (a extract) as described by Rasmussen et al. (1990). Rheumatoid factor (RF) was determined by a latex fixation test (Behringwerke, Marburg, F.R.G.). IgG was prepared from C-ANCA + sera by ammonium sulfate precipitation and ion exchange chromatography on DEAE-Sephadex (Pharmacia, Uppsala, Sweden). To minimize nonspecific fluorescent staining and
vals ranging from 5 to 300 min and fixed thereafter with ethanol.
Preparation of SK-RCll extracts SK-RCll cells were grown as described above in cell culture flasks at a cell density of 4 x 104 cells/cm 2. 108 cells were harvested after trypsin treatment (0.5 m g / m l for 5 min) by centrifugation at 800 x g for 5 min, washed with PBS and resuspended in 2 ml PBS containing 1 mM protease inhibitor phenyl-methyl-sulfonyl-fluoride (PMSF). Cell extracts were obtained by homogenization of the cells after freezing and thawing using a Dounce Potter homogenizer with an Spestle (30 strokes). Cell debris was removed by centrifugation (10,000 X g for 30 min at 4° C) and the supernatant used as a total extract. Antibody testing Antinuclear antibodies (ANA) were detected using IFT on rat tissue and HEp2 cells. HEp2 cells were commercially prepared (Antibodies, Davis, CA) and stained tissues were analyzed with a Zeiss Axiophot microscope equipped with epifluorescence optics (Zeiss, Oberkochen, F.R.G.). A super-pressure mercury lamp (HBO 50) served as a light source. Control experiments revealed that an excitation filter BP 485/20 and a cut-off filter LP 520 were specific for the fluorochrome dye used (data not shown). In addition, a Zeiss LSM 10 laser scan microscope (Zeiss, Oberkochen, F.R.G.) was employed and the reTABLE I ANTIBODIES DETECTED WITH DIFFERENT METHODS Diagnosis
Number of patients
Antibodies detected
IFT rat tissue
IFT HEp
IFT SK-RC11
Crit lucil.
CIE
Farr PEG
Neutr. Gran.
ANCA ELISA
WG SLE Scleroderma SS PBC MCTD RA(RF +) HBD
50 23 20 8 11 3 49 136
ANCA, Gol, ANA dsDNA, La Nucl, Ro, Gol Ro/La AMA, SMA ANA -
0 23 5 2 9 2 0 0
3 23 16 7 11 3 0 0
39 23 20 8 11 3 4 0
0 23 0 0 0 0 0 0
0 5(La) 4(Ro) 8 0 0 0 0
0 23 0 0 0 0 0 0
36 0 0 0 0 0 0 0
34 0 0 0 0 0 0 0
Key: WG, Wegener's granulomatosis; SLE, systemic lupus erythematosus; SS, Sj6gren's syndrome; PBC, primary biliary cirrhosis; MCTD, mixed connective tissue disease; RA, rheumatoid arthritis; HBD, healthy blood donors; ANA, anti-nuclear antibodies; dsDNA, antibodies to double stranded DNA; Nucl, antibodies to nucleoli; Gol, antibodies to the Golgi region; AMA, antimitochondrial antibodies; SMA, smooth muscle antibodies.
60
A
29
13
C
w,
i
Fig. 2. PAGE and Western blot. A: affinity purification usm a SK-RCll extract and a high titre C-ANCA + serum of WG patient; B: reaction of two C - A N C A + sera in a Western blot (29 kDa MW); C: reaction of a healthy blood donor (control) in a Western blot.
to e x c l u d e Fc r e c e p t o r b i n d i n g , ( F a b ' ) 2 f r a g m e n t s w e r e p r e p a r e d as d e s c r i b e d b y G a r v e y e t al. (1977). F i x e d S K - R C l l c e l l s w e r e i n c u b a t e d w i t h patients' sera or purified IgG fractions diluted
Fig. 1. IFT with a monospecific C-ANCA ÷ serum of a patient with WG (titre 1/640) (original magnification 630×). A: reaction on human neutrophils; B: reaction on SK-RCll cells; C: weak reaction on SK-RCll cells after preabsorption of antibodies with purified proteinase 3; D: weak reaction on human neutrophil granulocytes after preabsorption of antibodies with an extract of SK-RCll cells.
61 1 / 4 0 in PBS for 1 h in a humid c h a m b e r at room t e m p e r a t u r e . A f t e r extensive washing with PBS the cells were incubated with the second antibody, fluorescein isothiocyanate ( F I T C ) - c o n j u g a t e d anti-human I g G (F-5512, Sigma, Deisenhofen). Sections were then washed again and
incubated with Mowiol (Mowiol 4-88, H6chst, F r a n k f u r t F.R.G.). Live cells were stained by adding 10 #1 of undiluted serum or purified l g G fraction to the medium. Cells were washed with PBS and fixed immediately. M H C II expression was tested by incubation with a monoclonal anti-
Fig. 3. IFT on SK-RCll tumor cells (original magnification 630 x). A: reaction of a monospecificC-ANCA+ serum of a patient with WG (titre 1/640); B: reaction of 29 kDa antibody eluted from nitrocellulose (see Fig. 2); C: reaction of a monoclonal antibody to proteinase 3 (WGM2).
62 D R antibody (Becton Dickinson, Mountain View, CA). Affinity purified C - A N C A antigen was prepared as previously described (Liidemann et al., 1988) using an extract of human neutrophils or S K - R C l l cells. Proteinase 3 was purified as described by Kao et al. (1988). Polyacrylamide gel electrophoresis ( P A G E ) and Western blotting were performed as described previously (Bachmann et al., 1986; Mayet et al., 1988). For inhibition experiments serum of a C - A N C A + patient (1:640) was mixed v / v with affinity purified CA N C A antigen diluted to 0.1 m g / m l protein concentration in PBS or purified proteinase 3 and incubated on a rotator for 1 h at 3 7 ° C and overnight at 4 o C. The mixture was centrifuged at 30,000 × g for 15 min at 4 ° C and the supernatants kept as absorbed sera. A murine monoclonal antibody (mAb) to proteinase 3 designated W G M 2 (Csernok et al., 1990) was used as a control in the IFT.
Elution of antibodies from nitrocellulose Antibodies were eluted from nitrocellulose as described by McHugh et al. (1990) with minor modifications. Briefly, a probed strip of nitrocellulose with the band at 29 kDa was lined up accurately against an untreated native strip. A band was cut from this native strip corresponding to the area of reactivity on the stained strip. A band corresponding to an unstained area on the probed strip was also cut from the native sheet to act as a negative control. The bands were cut into 3 mm 2 pieces and placed into separate Eppendorf tubes. Each tube was filled with P B S / 0 . 0 5 % Tween (pH 7.3) and gently agitated for 10 min. This was followed by two washes with PBS and a
Fig. 4. Time-dependent antigen translocation after treatment of SK-RCll tumor cells with cytokines (IFN, TNF). IFT on fixed cells (original magnification 630 x ). A: weak cytoplasmic fluorescence and perinuclear concentration after 30 min of cytokine treatment; B: perinuclear concentration and weak nuclear fluorescence after 60 min of cytokine treatment; C: bright nuclear fluorescence after 75 rain of cytokine treatment; D: cytoplasmic redistribution after 135 min of cytokine treatment; E: reaction with monoclonal antibody to proteinase 3 (WGM2) after 120 min of cytokine treatment; F: incubation with a monospecific C-ANCA + serum after preabsorption of antibodies with purified proteinase 3: no reaction.
63
Fig. 5. Time-dependent antigen translocation after treatment with cytokines (IFN, TNF). IFT on fixed (A, B, D) and live (C) SK-RCll2 tumor cells; laser scanning microscopy; n = nucleus (original magnification 1000 x). A: weak nuclear fluorescence and perinuclear concentration; B: nuclear fluorescence; C: membrane expression; D: reaction with secretory vesicles leaving the trans-region of the Golgi complex.
wash with 0.25 M KC1 in PBS. After the last wash, 0.5 ml of the elution buffer (0.2 M glycine, p H 2.8) containing 1 m g / m l bovine serum albu-
min (BSA) was added to each tube and the contents agitated for 2 min. 1 M Tris was added to bring the p H back to neutral, the nitrocellulose
64
pieces were stored for further use and the eluates transferred to dialysis tubing for dialysis against large volumes of cold PBS.
Results
Antibodies detected by IFT on SK-RC11 cells recognized different nuclear and cytoplasmic antigens, as shown in Tables I and II. Staining patterns of antibodies of patients with LE, SS, scleroderma, MCTD and PBC were similar to the patterns obtained using rat tissue or HEp2 cells. Compared to standard procedures, sensitivity with S K - R C l l cells was slightly increased, especially for ANA (see also Table I). The most striking and unexpected result was that C-ANCA + sera of W G patients displayed a unique and characteristic diffuse cytoplasmic staining pattern (Fig. 1B). Using cultured cells antibody titres were comparable to the titres obtained with human granulocytes. There was no difference between ANCA + serum or a purified IgG fraction and modification of the fixation procedure (time, methanol, ethanol or paraformaldehyde) did not alter the binding pattern. C-ANCA + sera were almost negative on SKR C l l cells or human neutrophils when preabsorbed by incubation with an extract of human neutrophil granulocytes, S K - R C l l cells or purified proteinase 3 (Figs. 1C and 1D). A well
TABLE II ANTIBODIES D E T E C T E D BY IFT ON S K - R C l l T U M O R CELLS Diagnosis
WG
Number of patients 50
SLE 23 Sclerod. 20 SS 8 PBC 11 MCTD 3 R A (RF + ) 49 HBD 136
IFT-pattern on S K - R C l l cells
36 diff. cytoplasm, 21 Golgi, 5 ANA (2 homo, 3 speckled) 23 A N A (18 homo, 5 speckled) 20 A N A (20 nucleolar) 8 A N A (speckled) 11 AMA, 3 SMA 3 A N A (1 speckled, 2 centromer) 4 SMA 0
Fig. 6. IFT on live unfixed S K - R C l l cells incubated with purified IgG fraction of a C-ANCA + patient (original magnification 630 ×). Membrane expression of the antigen (arrow).
characterized mAb to proteinase 3 (WGM2) also produced the diffuse cytoplasmic staining pattern (Fig. 3C). Following affinity purification using a SK-RC11 extract and a high-titre C-ANCA + serum from a WG patient a protein of 29 kDa MW was isolated (Fig. 2A). This protein was recognized by C-ANCA + W G sera in a Western blot (Fig. 2B). Bound antibodies could be eluted from the nitrocellulose and again revealed the typical diffuse cytoplasmic staining pattern when performing IFT on S K - R C l l (Fig. 3B). Even after several passages in cell culture no changes in antigen expression could be observed. Treatment of live cells during culture with IFN and T N F led to a time-dependent translocation of the antigen (Figs. 4 and 5). After 30-45 min the antigen was concentrated in the perinuclear region and 30 min later it was found in the nucleus. The redistribution of antigen into the cytoplasm started 60 min later. After 120-240 min the antigen was detected on the surface of live cells (Fig. 6) colocalized with MHC II (DR) (data not shown). Similar staining patterns were seen after incubation with C-ANCA + sera or WGM2 (Fig. 4E). In addition, 21 of the 50 WG sera and five of the 20 scleroderma sera were shown to possess antibodies to cytoplasmic or-
65 ganelles, presumably secretory vesicles leaving the trans-region of the Golgi complex (Fig. 5D).
Discussion
In the early 80s, several groups independently reported the occurrence of antibodies directed against cytoplasmic antigens of neutrophils (ANCA) in patients with vasculitis and glomerulonephritis (Davies et al., 9182; Hall et al., 1984; Van der Woude et al., 1985). Subsequently, ANCA have proved to be a useful clinical tool in supporting the diagnosis and monitoring disease activity in WG (N611e et al., 1989; Cohen-Tervaert et al., 1990). Human neutrophil granulocytes and the promyelocytic HL-60 cell line have represented the exclusive sources of antigen for detecting autoantibodies in WG sera since studies have failed to reveal proteinase 3 in cells other than neutrophils and monocytes (Braun et al., 1991). Crude extracts of granulocytes have been used to characterize the relevant antigens by affinity chromatography with high titre ANCA + sera or monoclonal antibodies (Goldschmeding et al., 1988, 1990; Daha et al., 1990; Liidemann et al., 1990). Moreover, ANCA may play a role in pathogenesis by releasing proteolytic enzymes and O 2 radicals from neutrophil granulocytes (Falk et al., 1990). In this study we have tested 50 sera of patients with WG by performing IFT on a human renal cancer line SK-RCll and have found antibodies against nuclear and cytoplasmic antigens. In 39 patients C-ANCA + sera reacted with a characteristic diffuse cytoplasmic pattern on tumor cells and antibody binding could be inhibited by preabsorption with purified proteinase 3. Treatment of cells with cytokines led to a translocation of the antigen and this was further confirmed by cross-absorption experiments to exclude reactions with a different up-regulated nuclear antigen. The mechanism of this time-dependent translocation of the C-ANCA antigen triggered by cytokines such as IFN remains unclear. A similar antigen shuttling has recently been shown for the La/SS-B antigen in CV-1 cells (Bachmann et al., 1989). The antigen may be cotrans-
ported together with other autoantigens and membrane expression of the antigen could mediate the recognition of a cytoplasmic antigen by the host thereby enabling circulating autoantibodies to bind to these cells in vivo. Recently, we have demonstrated by immunoelectron microscopy the presence of C-ANCA antigen proteinase 3 in small amounts on the plasma membrane of resting normal human neutrophils and monocytes (Csernok et al., 1990). In contrast to the present results, we did not see membrane staining by IFT on unstimulated SK-RCll cells. However, it should be pointed out that immunoelectron microscopy is more sensitive than IFT and this may explain the different findings. Our preliminary data do at least hint at a possible functional role of the antigenic protein. Inhibition experiments with a crude extract of granulocytes, SK-RCll cells and purified proteinase 3 showed identity between the antigen recognized in neutrophils and SK-RCll. Antigenic identity was further confirmed by IFT using a mAb to proteinase 3. Proteinase 3 was described by Baggiolini et al. in 1978 as a result of observations made after nondenaturing PAGE of an extract of azurophilic granules from polymorphonuclear leucocytes (PMN) (Baggiolini et al., 1987). It is a neutral serine proteinase and has recently been shown to be important in the pathogenesis of human emphysema (Kao et al., 1988). Proteinase 3 is identical with myeloblastin, a serine protease whose complementary DNA has been cloned from promyelocytic leukaemia HL-60 cells. It appears to possess multiple biological activities, microbicidal and elastinolytic functions and a regulatory function during myelomonocytic cell differentiation (Jenne et al., 1990). This is, to our knowledge, the first report of the expression of this serine proteinase in nonleukemic human tumor cells. The antigen repertoire in SK-RC11 tumor cells seems to overlap with that of human neutrophils (Fig. 7). Despite the apparent differences between active tumor cells and neutrophils there are several common functional aspects. In order to pass through the blood vessel walls neutrophils adhere to the endothelial cells, squeeze between the ceils and through the basement membrane. The azurophilic granules of neutrophils are equivalent
66
Fig. 7. Comparison of IFT pattern on human neutrophil granulocytes and on SK-RC11 tumor cells (original magnification 630 x ). A: C-ANCA + serum of a WG patient on human granulocytes; B: C-ANCA + serum as used in A on S K - R C l l tumor cells (note characteristic diffuse cytoplasmic fluorescence and additional reaction with Golgi organelles); C: C-ANCA serum of a WG patient on human granulocytes; D: C - A N C A - serum as used in C on S K - R C l l tumor cells (note bright nucleolar fluorescence despite negative reaction on human granulocytes).
67 to t h e l y s o s o m e s in o t h e r cell types, i.e., t h e y c o n t a i n acid h y d r o l a s e s . O b v i o u s l y t h e s e e n z y m e s c a n cleave a large n u m b e r o f d i f f e r e n t tissue c o n s t i t u e n t s which a r e i m p o r t a n t for t h e struct u r e of t h e e x t r a c e l l u l a r m a t r i x o f c o n n e c t i v e tissue a n d for the s h a p e , m i g r a t i o n , g r o w t h a n d d i f f e r e n t i a t i o n o f cells ( R o o s et al., 1990). T h e invasion o f n o r m a l tissues a n d t h e p e n e t r a t i o n of v a s c u l a r b a s e m e n t m e m b r a n e s by m e t a s t a t i c tum o r cells m a y also r e q u i r e t h e active p a r t i c i p a tion o f hydrolytic enzymes. L y s o s o m a l c a t h e p s i n s have b e e n r e p o r t e d l y e l e v a t e d in s o m e t u m o r tissues a n d e n h a n c e d p r o d u c t i o n a n d s e c r e t i o n o f s e r i n e p r o t e a s e s have b e e n a s s o c i a t e d with t h e n e o p l a s t i c t r a n s f o r m a t i o n o f a v a r i e t y of cell t y p e s ( F i d l e r et al., 1989). T h e r e is clearly m u c h m o r e to b e l e a r n e d a b o u t t h e physical a n d f u n c t i o n a l p r o p e r t i e s o f p r o t e i n a s e 3. T h e s e investigations d e m o n s t r a t e t h a t the r e p e r t o i r e o f a n t i g e n s r e c o g n i z e d by a n t i b o d i e s in W G s e r a is not l i m i t e d to h u m a n n e u t r o p h i l s a n d m o n o c y t e s . This fact a g a i n p o i n t s to an a u t o i m m u n e p r o n e c o n d i t i o n in p a t i e n t s with W G (Shilitoe et al., 1974; G r o s s , 1989) a n d suggests a closer r e l a t i o n s h i p b e t w e e n W G a n d o t h e r connective tissue diseases. F u r t h e r i n v e s t i g a t i o n s will b e n e c e s s a r y to evaluate the production and secretion of prot e i n a s e 3 by t u m o r cells a n d to assess t h e diagnostic v a l u e o f I F T on d i f f e r e n t t u m o r cell lines in W G . I n a d d i t i o n , t h e d i f f e r e n t n u c l e a r antigens a n d c y t o p l a s m i c s t r u c t u r e s r e c o g n i z e d by W G s e r a a n d t h e i r m e m b r a n e e x p r e s s i o n n e e d to be c h a r a c t e r i z e d in m o r e detail.
Acknowledgements W e a r e g r a t e f u l for t h e t e c h n i c a l a s s i s t a n c e f r o m Mrs. A. Canisius, Ms. S. Mayer, Ms. J. K a r b a c h a n d Mrs. Sayah. W e wish to t h a n k Professor Dr. M. B a c h m a n n , I n s t i t u t fiir Physiologische C h e m i e , Universit~it M a i n z , for h e l p f u l discussions a n d e x p e r t t e c h n i c a l advice c o n c e r n i n g laser scanning microscopy. We thank Professor Dr. H. L e h m a n n , E v a n g e l i s c h e s K r a n k e n h a u s Z w e i b r i i c k e n , 6660 Z w e i b r i i c k e n , F . R . G . , for p e r mission to r e p o r t p a t i e n t s u n d e r his care.
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