ANTICYTOPLASMIC ANTIBODIES AND WEGENER'S GRANULOMATOSIS
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1 J P^ Cynthia Ml Iler, M.D, September 1989 ANTICYTOPLASMIC ANTIBODIES AND WEGENER'S GRANULOMATOSIS Background Wegener's granulomatosis, a disease of unknown etiology, is characterized by a granulomatous vasculitis of the upper and lower r e s p i r a t o r y t r a c t s. Va r y i n g d e g r e e s o f s y s t e m i c v a s c u l i t i s o f s m a l l arteries and veins may also be seen <1>. Renal disease in the form of f o c a l o r d i f f u s e n e c r o t i z i n g g l o m e r u l i t i s i s o f t e n p r e s e n t. U n t i l r e c e n t l y, t h e d i s e a s e w a s u n i f o r m l y f a t a l. To d a y, I m m u n o s u p p r e s s i v e and cytoxic therapy are used with up to 90% of patients responding <2). The disease is diagnosed by biopsy of affected tissue, preferably l u n g. U n f o r t u n a t e l y, o b t a i n i n g a g o o d s p e c i m e n I s o f t e n d i f fi c u l t o r i m p o s s i b l e. E v e n w h e n a b i o p s y i s o b t a i n e d, a d e fi n i t i v e d i a g n o s i s cannot always be made. In many cases, diagnosis and disease activity must be based on clinical impression and nonspecific lab tests such as ESR and CRP (3). In 1985, Van Der Woude, et al., first described an ant 1 cytoplasmic antibody <ACPA) which they felt to be an autoantibody s p e c i fi c f o r We g e n e r ' s g r a n u l o m a t o s i s ( 4 ). S i n c e t h e n, n u m e r o u s reports have validated the association between Wegener's granulomatosis and ACPA. Although both RIA <7,10) and ELISA (12,13,20) methods have been developed, ACPA is most commonly detected A f b y i n d i r e c t i m m u n o fl u o r e s c e n t a s s a y s.
2 p e r i n u c l e a r s t a i n i n g a s w e l l a s t h e d i f f u s e c y t o p l a s m i c s t a i n i n g s a i d Methodology ACPA measurements are not yet commerlcally available. Thus, there are many variations on the test methodology depending on the group that performs them. However the basic principle of the assays remains the same. These tests allow for detection of ACPA by applying p a t i e n t s e r u m t o a p r e p a r a t i o n o f g r a n u l o c y t e s. F l u o r e s c e n t a n t i - h u m a n I g G i s a d d e d, a l l o w i n g v i s u a l i z a t i o n o f a n t i b o d y w i t h i n t h e granulocytes. The following is the method of Van Der Woude, et al., the group that first developed the assay (6). C ytocentrifuge slides o f g ranulocytes a re p repared from peripheral blood of normal human controls. Dextran sedimentation and l y s i s o f r e d b l o o d c e l l s w i t h d i s t i l l e d w a t e r y i e l d a c l e a n p r e p a r a t i o n o f c e l l s. T h e s e c e l l s a r e fi x e d t o g l a s s s l i d e s w i t h 9 5 % ( e t h a n o l a t 4 C. T h e s l i d e s a r e i n c u b a t e d w i t h p a t i e n t s e r a f o r 3 0 minutes, then washed with phosphate buffered saline <PBS>. Next, the slides are incubated with Fluorescein Isothlocyanate conjugated sheep anti-human IgG for 30 minutes,then washed. Slides are evaluated under a fl u o r e s c e n t m i c r o s c o p e. A C PA i s c h a r a c t e r i z e d b y s t r o n g, d i ff u s e s t a i n i n g o f t h e e n t i r e c y t o p l a s m, l e a v i n g t h e n u c l e u s u n s t a i n e d. Discussion A number of studies have now been published proposing ACPA as a m a r k e r f o r We g e n e r ' s G r a n u l o m a t o s i s ( 4, 5, 6, 11, 2 0 ). M o s t c l a i m a v e r y high specificity. The largest study, including 1657 controls, claims a s p e c i fi c i t y o f % f o r I FA. M o s t o f t h e f a l s e p o s i t i v e s i n t h e m a j o r i t y o f t h e s e s t u d i e s w e r e d u e t o o t h e r u n c l a s s i fi e d v a s c u l i t i d l e s. T h e h i g h s p e c i fi c i t y i s h i g h l y d e p e n d e n t o n g o o d technique and experienced interpretation of the fluorescent patterns. S e v e r a l s t u d i e s, s h o w i n g c o n s i d e r a b l y l o w e r s p e c i fi c i t y, c o u n t e d
3 overlap In specificity noted by some groups. t o b e s p e c i fi c f o r We g e n e r ' s g r a n u l o m a t o s i s ( 1 3, 1 4, 1 7, 1 8 ). I t i s o n l y ^ w h e n p o s i t i v e s a r e r e s t r i c t e d t o t h e d i f f u s e c y t o p l a s m i c s t a i n i n g t h a t h i g h s p e c i fi c i t y i s o b t a i n e d. A n u m b e r o f c a s e r e p o r t s a r e n o w i n t h e literature supporting ACPA as a specific marker of Wegener's g r a n u l o m a t o s i s ( 1 5, 1 6, 1 9 ). O t h e r c a s e r e p o r t s, c o n t r a d i c t t h e s e fi n d i n g s ( 9, 11, 1 8 ). H o w e v e r, m a n y o f t h e s e l a t t e r c a s e s I n v o l v e d i s e a s e s, s u c h a s m i c r o s c o p i c p o l y a r t e r i t i s, w h i c h a r e i l l - d e fi n e d entities. These may in fact be related to Wegener's granulomatosis. The sensitivity of the ACPA test varies considerably from 56% to 96% depending on what group of patients is included (4,5,6,11,20). When all patients with Wegener's granulomatosis are counted, the sensitivity ranges from 56% to 76%. The sensitivity rises to 96% when r e s t r i c t e d t o p a t i e n t s w i t h a c t i v e g e n e r a l i z e d d i s e a s e. T h i s f a c t h a s led some people to conclude that the test can be used as a marker of disease activity. S e v e r a l p o i n t s w e a k e n t h i s c o n c l u s i o n. I n m o s t o f t h e s t u d i e s, t h e m a j o r i t y o f p a t i e n t s w e r e d e fi n e d b y b i o p s y d i a g n o s i s. H o w e v e r, a n u m b e r o f p a t i e n t s w e r e d e fi n e d b y c l i n i c a l p a r a m e t e r s a l o n e. T h i s c a s t s s o m e d o u b t o n t h e r e l i a b i l i t y o f t h i s t e s t ' s s e n s i t i v i t y. I n addition, most of these studies used nonspecific markers such as CRP and ESR to define disease activity. I t i s n o t y e t c l e a r w h a t t h e a n t i c y t o p i a s m l c a n t i b o d y i s d i r e c t e d a g a i n s t. R e s e a r c h e r s h a v e n o t e d a n a s s o c i a t i o n w i t h a l k a l i n e Phosphatase (7) and with myeloperoxidase (13). However, both of these claims have been disputed (8,12,14). The antibody has most f0j$^t\ c o n s i s t e n t l y b e e n a s s o c i a t e d w i t h t h e s p e c i fi c g r a n u l e o f t h e n e u t r o p h i l, b u t a s p e c i fi c p r o t e i n h a s n o t b e e n i d e n t i fi e d ( 8, 1 2 ). I n d e e d, s o m e f e e l t h a t t h e a n t i b o d y i s a c t u a l l y d i r e c t e d a g a i n s t a h e terogeneous m i x o f p r o teins ( 1 7 ). This w o u l d a c c o u n t for the
4 Most groups have used the IFA technique to study the ACPA. This is the easiest and most reproducible method. In an effort to overcome ( t h e p r o b l e m s I n h e r e n t t o t h i s m e t h o d, a n u m b e r o f g r o u p s h a v e developed RIA and ELISA assays (7,10,12,13,20). However, these methods have their own shortcomings. Most Important is the varying s p e c i fi c i t y o f t h e s e t e s t s. T h i s i s d u e t o t h e d i f f e r i n g m e t h o d s o f preparing the antigen extract used by each group. As yet none of t h e s e a s s a y s i s w i d e l y a v a i l a b l e. In summary, Wegener's granulomatosis is a uniformly fatal disease w i t h o u t t r e a t m e n t. T h e d i a g n o s i s i s o f t e n q u i t e d i f fi c u l t, e v e n w i t h a t i s s u e b i o p s y, a n d p a t i e n t s a r e o f t e n t r e a t e d w i t h o u t a d e fi n i t i v e d i a g n o s i s. U n f o r t u n a t e l y, s o m e o f t h e d i s e a s e s i n t h e d i f f e r e n t i a l d i a g n o s i s a r e t r e a t e d q u i t e d i f f e r e n t l y. T h u s a t e s t s p e c i fi c a l l y f o r Wegener's granulomatosis would be highly desirable. The a a n t i c y t o p l a s m i c a n t i b o d y ( A C PA ) i s a n i n t r i g u i n g p o s s l b l 1 i t y. I t appears to be quite specific, though not very sensitive for Wegener's g r a n u l o m a t o s i s. T h e a b i l i t y o f t h i s a s s a y t o f o l l o w d i s e a s e a c t i v i t y is still unclear. The antigen measured by this assay remains unknown. Although ACPA is clearly not ready to replace tissue biopsy as the g o l d s t a n d a r d f o r d i a g n o s i s o f We g e n e r ' s g r a n u l o m a t o s i s, i t c l e a r l y d e s e r v e s f u r t h e r s t u d y. References 1. F a u c i, A., e t a l., " We g e n e r ' s g r a n u l o m a t o s i s : P r o s p e c t i v e c l i n i c a l and therapeutic experience with 85 patients for 21 years", Annals of Internal Medicine, 1983;98: JltlfeN, 2. C o t r a n, R., e t a l., R o b b i n s P a t h o l o g i c b a s i s o f d i s e a s e, 4 t h edition, W.B.Saunders, Sack,K., "Wegener's granulomatosis", Western J of Medicine, 1 QflQ ; 1 RO "50Q
5 J0fev 4. Van Der Woude,F., et al., "Autoantibodies against neutrophils and monocytes: Tool for diagnostic and marker of disease activity in Wegener's granulomatosis", Lancet, 1985;1: G r o s s, W., e t a l., " A n t i c y t o p l a s m i c a n t i b o d i e s i n We g e n e r ' s granulomatosis"(letter), Lancet, 1986;1: Ludemann,G., and Gross,W., "Autoantibodies against cytoplasmic s t r u c t u r e s o f n e u t r o p h i l g r a n u l o c y t e s i n We g e n e r ' s g r a n u l o m a t o s i s ", Clin. exp. Immunol., 1987;69: Lockwood,C, et al., "Association of alkaline phosphatase with an a u t o a n t i g e n r e c o g n i s e d b y c i r c u l a t i n g a n t i - n e u t r o p h i 1 a n t i b o d i e s i n s y s t e m i c v a s c u l i t i s ", L a n c e t, ; 1 : r 8. R a s m u s s e n, N., e t a l., A n t i - n e u t r o p h i 1 c y t o p l a s m a n t i b o d i e s i n Wegener's granulomatosis are not directed against alkaline phosphatase"(letter), Lancet, 1987;1: Wathen,C, and Harrison,D., "Circulating anti-neutrophi1 antibodies i n s y s t e m i c v a s c u l i t i s " ( L e t t e r ), L a n c e t, ; 1 : Savage,C., et al., "Prospective study of radioimmunoassay for a n t i b o d i e s a g a i n s t n e u t r o p h i l c y t o p l a s m i n d i a g n o s i s o f s y s t e m i c vasculitis", Lancet, 1987;1: Ve n n i n g, M., e t a l., " A n t i b o d i e s t o n e u t r o p h i l c y t o p l a s m i c a n t i g e n i n s y s t e m i c v a s c u l i t i s " ( L e t t e r ), L a n c e t, ; 2 : Ludemann,J., et al., "Detection and quantitation of antl-neutrophl1 cytoplasm antibodies In Wegener's granulomatosis by ELISA using affinity-purified antigen", J. of Immun. Methods, 1988;114:
6 13. Falk,R., and Jennette,J., "Anti-neutrophl1 cytoplasmic ^ autoantibodies with specificity for myeloperoxidase In patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis", N Engl J Med, 1988;318: Specks,U., et al., "Anti-neutrophi1 cytoplasmic autoantibodies"(letter), N Engl J Med, 1988;319: Hoare,T., and Rhys Evans,P., "Anti-neutrophi1 cytoplasmic antibody assay in diagnosis of recurrent subglottic stenosis"(letter), Lancet, 1988;2: Pudlfin.D., et al., "Circulating auto-antibodies to neutrophil cytoplasmic antigens in vasculitis", S African Med J, 1989;75: Thompson,R., and Lee, S., "Antineutrophl1 cytoplasmic (^ antibodies"(letter), Lancet, 1989;1: Nassberger,L., et al., "Circulating anti-neutrophi1 cytoplasm antibodies in patients with rapidly progressive glomerulonephritis and extracapillary proliferation", J. of Int Med, 1989;225: Feehally,J., et al., "A case of microscopic polyarteritis with antineutrophl1 cytoplasmic antibodies"(letter), Clin Nephrol, 1987;27: Nolle,B., et al., "Ant 1 cytoplasmic autoantibodies: Their immunodiagnostic value in Wegener's granulomatosis", Ann of Int Med, 1989;111:28-40.
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