This thesis studies the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in idiopathic inflammatory disorders in which GS-ANA were described during the 60s: rheumatoid arthritis, autoimmune liver diseases, inflammatory bowel disease, and juvenile chronic arthritis. In all of these disorders the presence of ANCA was related to disease activity, disease duration and severity of disease. Furthermore, the antigenic specificity of ANCA in chronic inflammatory disorders was studied. ... Zie: Summar

Mulder, Alide Heleen Leontine

University of Groningen

English

Anti-neutrophil cytoplasmic antibodies in idiopathic inflammatory disorders

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Anti-neutrophil cytoplasmic antibodies in idiopathic inflammatory disorders
Mulder, Alide Heleen Leontine
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Publication date:
1994
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Mulder, A. H. L. (1994). Anti-neutrophil cytoplasmic antibodies in idiopathic inflammatory disorders s.n.
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This thesis studies the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in
idiopathic inflammatory disorders in which GS-ANA were described during the 60s:
rheumatoid arthritis, autoimmune liver diseases, inflammatory bowel disease, and juvenile
chronic arthritis. In all of these disorders the presence of ANCA was related to disease
activity, disease duration and severity of diseae. Furthermore, the antigenic specificity of
ANCA in chronic inflammatory disorders was studied. In addition, this thesis investigated
the pathophysiological significance of ANCA in chronic inflammatory disorders, by
studying the capacity of ANCA to activate granulocytes.
Chapter f is a review in which target antigens of ANCA and the clinical significance of
the different ANCA specificities are discussed. The classical or cytoplasmic ANCA (c-
ANCA) is in almost all of the cases directed against proteinase 3. C-ANCA occur in more
than 90% of patients with extended Wegener's granulomatosis (WG), n 75% of patients
with limited WG, and in some 40 ta 50% of patients with vasculitic overlap syndromes.
The presence of c-ANCA is highly specific for those diseases and changes in levels of c-
ANCA precede disease activity. Perinuclear ANCA (p-ANCA) are directed against
different cytoplasmic constituents of neutrophils and occur in a wide range of diseases. p-
ANCA directed against myeloperoxidase have a high specificity for necrotizing vas-
culitides, but may incidentally occur in other diseases as well. p-ANCA directed against
elastase are incidentally found in patients with vasculitic disorders, whereas lactoferrin
antibodies are detected in patients with primary sclerosing cholangitis with or without
ulcerative colitis, and in rheumatoid arthritis. p-ANCA of undefined specificity can be
detected in ulcerative colitis, Crohn's disease, autoimmune liver diseases, chronic
arthritides and in some 5% of healthy controls. The diagnostic value of p-ANCA of
undefined specificity has to await characterization of the antigens involved.
Chapter 2 studies the prevalence, interrelationships and target antigens of ANCA in
rheumatoid arthritis, and relates the presence of ANCA to disease duration and occurrence
of extra-articular manifestations. ANCA were detected in 7O% of rheumatoid sera, wheras
in 36% of the sera the antibodies reacted with cytoplasmic constituents of the neutrophil as
shown by a cytoplasmic staining pattern on paraformaldehyde fixed granulocytes. Elisa
studies showed that 20Vo of the sera reacted with lactofernn, lVo with myeloperoxidase,
nd lVo with elastase. Western blotting studies confirmed the Elisa data and revealed
reactivity with hitherto unknown polypeptides of 67166 kD (6Vo) and 63154 kD (9%).
Neither of the antibodies was associated with a particular subset of disease, but the
prevalence of the antibodies tended to increase with longstanding disease. Anti-lactoferrin
antibodies were demonstrated to be present in synovial fluid as well.
Chapter 3.1 studies the diagnostic significance of ANCA in chronic liver diseases by
testing sera from patients with primary biliary cirrhosis (PBC), primary sclerosing
cholangitis (PSC), autoimmune chronic hepatitis (AI-CAH) and non-autoimmune liver
diseases. ANCA were detected in 28% of PBC sera, in 79% of PSC sera, and in 88% of
AI-CAH sera, while they were not detected in non-autoimmune liver disease sera. The
presence of ANCA was significantly correlated with the presence of cirrhosis. The ANCA
antigens involve<i were not Pr3, MPO or HLE, but Western blotting studies showe<l
reactivity with lactoferrin, the 67166 kD doublet and a 40 kD protein. Reactivity with
either of these proteins was observed in sera from patients with PBC (2O%), PSC (38%)
and AI-CAH (17%).
Chapter 3.2 approaches the possible immunopathogenetic importance of ANCA in PSC by
126
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Chapter 4.1 st'
ANCA can be
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compared to C.
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Chapter 4.2 st
positive sera ir
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showed reactivi
46% of the UC
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suggest that Al
diverse conditi<
Chapter 5 desc
juvenile inflam
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ticular onset J(
No relation we
prolonged dise
frequently dete
disease activity
cytoplasmic fl
nuclear fluores
of patients wit
MPO or HLE
with lactoferrir
Chapter 6.1 st
nism. Primed
PR3, MPO anr
respective antr
production of r
depended on tl
receptors on tl
Not all ANCA
tlCA) in
the 60s:
juvenile
r disease
ificity of
estigated
lers, by
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els of c-
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' can be
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but the
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ases by
lerosing
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88Vo of
ra. The
ANCA
showed
ty with
(38%)
)SC by
studying the occurrence of ANCA in PSC before and after liver transplantation. Nine
patients with PSC, all positive for p-ANCA, showed a decline in ANCA titer immediately
after liver transplantation, but during follow-up ANCA titers equal to pre-transplantation
titers were measured. No recurrence of disease could be observed, as judged by liver
histology. Control patients were ANCA negative, and remained ANCA negative after liver
transplantation. This study demonstrates .hat ANCA remain present after liver transplan-
tation and that the synthesis of ANCA is not related to the presence of the diseased organ.
Chapter 4.1 studies the prevalence of ANCA in inflammatory bowel diseases (IBD). p-
ANCA can be demonstrated in 49% of patients with ulcerative colitis (UC), and n 40%
of patients with Crohn's disease (CD). Titers of ANCA are higher in patients with UC
compared to CD patients. The antigenic specificity of ANCA in IBDas tested by Elisa is
generally unknown, although incidental sera recognize lactoferrin or myeloperoxidase. So,
within the spectrum of IBD, the presence of p-ANCA is not specific for UC. When titers
of ANCA are taken into account, the presence of high+itered p-ANCA suggests active
UC.
Chapter 4.2 studies the antigenic specificity of ANCA in IBD. 76Vo of the p-ANCA
positive sera in UC and 50% of the p-ANCA positive sera in CD showed cytoplasmic
fluorescence on paraformaldehyde fixed neutrophils, indicating that indeed cytoplasmic
antigens are recognized by a considerable number of these sera. Western blot analysis
showed reactivity with either lactoferrin, the 67166 kD doublet or the 63/54 kD doublet in
46Vo of the UC sera and in 32% of the CD sera. Since identical patterns of reactivity have
been observed in rheumatoid arthritis sera and autoimmune liver disease sera these data
suggest that ANCA of restricted specificities are not specific for IBD but are present in
diverse conditions characterized by chronic infl ammation.
Chapter 5 describes the prevalence of ANCA in juvenile chronic arthritis (JCA) and other
juvenile inflammatory disorders such as cystic fibrosis, juvenile diabetes and connective
tisue diseases. p-ANCA are detected in 35Vo of JCA sera and in only 6Vo of the disease
control sera. Considering the onset type of JCA, ANCA are detected n 44Vo of oligoar-
ticular onset JCA, in 36Vo of polyarticular onset JCA, and in l6Vo of systemic onset JCA.
No relation was observed between ANCA and either the presence of rheumatoid factor,
prolonged disease or more progressive disease. However, ANCA were significantly less
frequently detected during remission of JCA suggesting that ANCA might be a marker of
disease activity. On paraformaldehyde fixed neutrophils only 14% of the JCA sera showed
cytoplasmic fluorescence, whtle 23Vo of the sera showed nuclear fluorescence. The
nuclear fluorescence pattern was related with the presence of ANA, especially in the sera
of patients with polyarticular onset JCA. Antigen specificity studies showed that PR3,
MPO or HLE are not the antigens involved. Western blotting studies showed reactivity
with lactoferrin (5Vo) or polypeptides of 67166 kD (9Yo).
Chapter 6..I studies neutrophil activation by ANCA, and analyzes the underlying mecha-
nism. Primed normal donor neutrophils are demonstrated to express the ANCA antigens
PR3, MPO and lactoferrin on the cell surface, and interaction of these antigens with their
respective antibodies resulted in activation of the neutrophils as demonstrated by the
production of superoxide. In contrast to other reports, activation of neutrophils by ANCA
depended on the presence of the Fc-part of the antibodies, and the availability of the Fc7-
receptors on the neutrophils. Especially the second Fc7-receptor appeared to be involved.
Not all ANCA positive samples were capable of granulocyte activation irrespective of the
tz'l
ANCA titer. Inlerestingly, samples capable of granulocyte activation contained relatively
high levels of the IgG3 subclass of ANCA in contrary to the ANCA positive samples that
could not induce activation.
Chapter 6.2 extends the studies of chapter 6.1. Paired ANCA positive samples from
active and inactive disease were studied for their capacity to induce the respiratory burst,
and granulocyie activation was related to ANCA titer and subclass distribution, together
with disease activity. Sera of patients with active disease better induced the respiratory
burst in primed neutrophils than sera of patients with inactive disease. There was a
significant relation between ANCA titer and the amount of superoxide produced. In
addition, changes in IgG3-subclass of ANCA appeared to correlate with changes in super-
oxide production. Therefore, not only changes in ANCA titer during the course of
disease, but also changes in levels of lgG3-subclass of ANCA are important as the latter
are directly related to the neutrophil activating capacity of the autoantibodies.
The final chapter of this thesis, chapter 7, is an overall discussion on the data generated
in this thesis. It concludes with the vicious circle of ANCA associated idiopathic inflam-
matory disorders: ANCA are capable of activation of primed neutrophils. This results in
respiratory burst and degranulation. The ANCA antigens are now availalbe for immune
complex formation and new antibodies can be produced.
With respect to the questions posed in chapter 1.2 we conclude:
1. ANCA in the idiopathic inflammatory disorders have comparable antigenic specificities,
in particular lactoferrin and polypeptide doublets of 67166 and 63154 kD, suggesting that
these ANCA are not specific for one disease entity. ANCA of those specificities rather
se€m a marker for inflammatory disorders on an autoimmune background.
2. ANCA in rheumatoid arthritis and autoimmune liver diseases are related to either
disease duration or severity of disease. This .relation is less clear for ANCA in the
inflammatory bowel diseases and juvenile chronic active hepatitis.
3. The pathogenetic role of ANCA remains in part to be elucidated. Nevertheless, ANCA
can activate primed neutrophils. This process is Fc-dependent and particularly exerted by
the IgG3 subclass of ANCA. These data suggest that ANCA are capable of maintaining or
even amplifying the inflammatory process.
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Anti-neutrophil cytoplasmic antibodies in idiopathic inflammatory disorders

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