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1 Department of Clinical Immunology and Rheumatology,Ankara University School of Medicine Ankara, Turkey
2 Department of Internal Medicine, Süleyman Demirel University School of Medicine, Isparta,Turkey
3 Department of Infectious Diseases and Clinical Microbiology, Kırıkkale University School of Medicine, Kırıkkale, Turkey
Correspondence Addresse:
Nurşen Düzgün M.D.
Dedekorkut sok. 22 / 5 A.Ayrancı 06550
ANKARA/TURKEY
Telephon number: +90 312 3103333/2063
Fax number: +90 312 3097779
e-mail: duzgun@medicine.ankara.edu.tr
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ABSTRACT |
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INTRODUCTION |
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MATERIAL AND METHODS |
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DISSCUSSION |
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ACKNOWLEDGMENTS |
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REFERENCES |
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ABSTRACT
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Anti neutrophil cytoplasmic antibody (ANCA) is strongly associated with some vasculitic disorders. Behçet’s disease (BD) is a systemic vasculitis of unknown etiology. In this study, ANCA was found to be positive in 8 out of 66 patients (10.2 %) with BD by combination testing consisting of immunofluorescence and ELISA [one patient showed an atypical pattern by indirect immunofloresence techique, 6 patients were reactive to bacterial-permeability increasing protein (BPI) and one patient was reactive to Cathepsin G in ELISA]. There were no vascular manifestations such as veneous or arterial thrombosis and arterial aneurysms in ANCA-positive patients with BD. The results suggest that ANCA may be found in a minority of BD as those in previous published studies.
KEY WORDS: ANCA. Behçet’s disease. vasculitis
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INTRODUCTION |
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Anti-neutrophil cytoplasmic antibodies (ANCA) are strongly associated
with some vasculitic disorders. In systemic vasculitis, interactions between
neutrophils and ANCA may initiate endothelial and vascular injury. The presence
of ANCA has been established as serological marker especially in the ANCA-associated
vasculitis such as Wegener granulomatosis, Churg-Strauss syndrome, microscopic
polyangiitis and renal–limited glomerulonephritis (1).
Behçet’s disease (BD) is a chronic, relapsing and multisystem inflammatory
disorder with mucocuteneous, ocular, vascular, articular, gastrointestinal and
central nerveous system manifestations of unknown etiology (2,3). The main
histological finding of BD is vasculitis both in small and large blood vessels.
Histopathology of superficial thrombophlebitis and deep vein thrombosis revealed
vasculitis infiltrated lymphocytes and neutrophils (4). The studies have also
shown the presence of leucocytoclasis and necrotizing vasculitis (5), increased
chemotaxis and phagocytosis (6) and crescentic glomerulonephritis (7) in BD.
Neutrophils play an important role in inflammatory response by overproduction of
monocyte derived TNF-α, IL-6 and IL-8 (8). Several studies investigated ANCA to
assess the role of polymorphonuclear leucocytes in BD (9-11).
By indirect immunofluorescence techique (IIFT) on ethanol-fixed neutrophils, at
least two major patterns of ANCA can be identified: first, the cytoplasmic
pattern (cANCA) which reflects the presence of anti-proteinase 3 (PR3)
antibodies and second, the perinuclear pattern (pANCA) which is mainly produced
by anti-myeloperoxidase (MPO) antibodies. PR3 and MPO are contained in
cytoplasmic granules and induce the development of ANCA. ANCA against other
antigens such as lactoferrin (LF), cathepsin G (Cat.G), elastase (EL), lysozyme
(LY) and bactericidal/permeability-increasing protein (BPI) can be identified by
ELISA (12). ANCA are mostly tested for their anti-PR3 and anti-MPO activities by
either IIFT alone or both by IIFT and ELISA.
We aimed at detecting the presence of ANCA and their clinical importance in the
patients with BD, via making an assessment of their reactivity to PR3, MPO and
other antigens including LF, Cat.G, EL, LY and BPI using both IIFT (pANCA, cANCA)
and ELISA.
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MATERIAL AND METHODS
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Study population
This study included 66 patients (29 females with the mean age of 33.51+/-
standard deviation (SD) 9.86 and 37 males with the mean age of 33.74+/- SD 9.91,
range 15-56 years ) and 20 healthy controls (13 females with the mean age of
28.51+/- 9.57 (SD) and 7 males with a mean age of 29.14+/-3.18 (SD), range 18-
53). All patients fulfilled the International Study Group criteria for the
diagnosis of BD (3). Disease duration was 5.7+/-4.8 years. Clinical symptoms of
the patients are seen on table 1. Thirteen patients were on prednisone, 5
patients were on combination of these and other patients were on colchicine.
Nine patients without symptom had no therapy.
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ANCA testing:IIFT
Antibodies against granulocytes (c-ANCA / p-ANCA) were analysed by IIFT using a
BIOCHIP combination of ethanol-fixed as well as formalin-fixed granulocytes and
primate liver in sixty-six sera samples from patients with BD and twenty sera of
healthy group. Biochip technology: thin glass slides coated with biological
substrates are cut into millimetre-sized fragments (biochips) on a machine.
Biochips containing ethanol-fixed human granulocytes are used as a standart
substrate for immunofluorescence. Since antibodies against cell nuclei can hide
a p-ANCA pattern, an other biochip containing form-aldehyde fixed granulocytes
is added, allowing the detection of a part of the p-ANCA, particularly
antibodies against MPO, whereas nearly all antinuclear antibodies are completely
suppressed. The differentiation between p-ANCA and antibodies against cell
nuclei in IIFT can be difficult in some cases, therefore a biochip coated with
primate liver is used as additional substrate. It is possible to identify the
nuclei of the hepatocytes and the granulocytes in the sinusoids: the nuclei of
the neutrophils fluoresce with much more intensity than the hepatocyte nuclei.
The following titers were regarded as positive : c-ANCA>1:32, p-ANCA>1:10.
ANCA testing :ELISA
These sera were tested for autoantibodies against PR3, MPO, LF, Cat.G, EL, LY
and BPI using ELISA. The ELISA test kit contains microtiter strips each with 8
reagent wells separately coated with these antigens. The first reaction
step:diluted patient samples were incubated with the wells. If the sample is
positive, specific antibodies bind to the antigens. A second step: the bound
antibodies were detected using peroxidase-labelled anti-human antibodies, which
gives a color reaction. The intensity of the formed colour is proportional to
the concentration of the corresponding antibodies. Results are shown as
“negative” or in “positive” cases, in RU/ml. The cut-off is 20 RU/ml.
Results
p-ANCA by IIFT was found to be negative in all sera whereas c-ANCA atypical
pattern was detected in one patient (1/66 in IFTT). The 6 of 66 sera had
antibodies against BPI , only one serum was positive for antibody against
cathepsin G by ELISA (7/66 in ELISA).Therefore ANCA positivity was 8/66 (10.2%)
in this study (Table 2) Antibodies against other granulocyte antigens were not
found by ELISA. Healthy control group was ANCA-negative both by ELISA and IIFT.
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| DISCUSSIONS
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The development of ANCA-positive small vessel vasculitis depends on an auto-
immune response. Once auto-antibodies develop, they may activate neutrophils and
monocytes and injure endothelial cells. Other stimuli such as infection or
environmental exposure are known to contribute to the induction of ANCA mediated
vessel inflammation (13).
The etiology of BD is still unknown and both viral and autoimmune mechanisms
have been proposed. Some immune abnormalities such as the functional aberration
of T-cell subsets and mild overactivity of B-cells (14,15) with the presence of
autoantibodies such as anti-endothelial cell antibodies and anti-cardiolipin
antibodies have been reported in BD (16,17). These antibodies may contribute to
the development of vascular damage and neutrophils are implicated in the
pathogenesis of BD (18).
ANCA have rarely been found in patients with BD. In a cohort of 28 patients with
BD, only one patient had ANCA-positive by IIFT (10). Our observation that only
one serum showed atypical ANCA-positivity by IIFT, is in line with previous
studies. Ben Hmida et al. did not find detectable ANCA in 46 active and inactive
patients with BD (11). ANCA associated vasculitis and renal failure was reported
in a female (19) and a male patient (20) with BD. In another study, ANCA were
evaluated by IIFT and ELISA for anti-PR3 and anti-MPO antibodies and ANCA were
found in three out of 29 patients (10%) , mainly those with vasculitis (21). The
results of the present study also show that the sera from 8 out of 66 patients
(10.2 %) had ANCA positivity (1 in IIFT , 7 in ELISA ) and that there were no
vascular manifestations in these patients. Six out of 7 patients were reactive
to BPI in ELISA. The patients showing BPI-ANCA positivity may have negative
IIFT-ANCA results (22-24) as those in our findings. Burrows et al. (10) have
previously described a patient with BD who exhibited the presence of antibodies
to BPI as also unusual cutaneous manifestations. This article reports for the
first time, some association of BD with anti-BPI ANCA. However, the presence of
BPI-ANCA has previously been reported in the case of chronic infections such as
chronic bronchitis or cystic fibrosis (22,23) and during chronic inflammation
such as inflammatory bowel disease (24).
Our data suggest that ANCA positivity may be found probably as a non-spesific
finding in a minority of the patients with BD.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the invaluable help we received from Dr.rer.nat.Wolfgang Schlumberger who provided us to perform this study in Euroimmun, Laboratorium für experimentelle Immunologie GmbH, Lübeck, Hamburg
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REFERENCES
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