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On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.79 no.1 Rio de Janeiro Jan./Feb. 2004
CLINICAL, LABORATORY AND THERAPEUTIC INVESTIGATION
Antineutrophil cytoplasmic antibody (ANCA) in pyoderma gangrenosum, a serologic marker for associated systemic diseases: a study of eight cases*
Virgínia Lúcia Ribeiro CabralI; Sender Jankiel MiszputenII; Wilson Roberto CatapaniIII
Degree in Gastroenterology, UNIFESP / EPM
IIAdjunct Professor of Gastroenterology, UNIFESP / EPM
IIITitular Professor of Gastroenterology, ABC Faculty of Medicine
The pathogenesis of Ulcerative Colitis (UC) and its extraintestinal manifestations
remain uncertain, although involvement of the immune system is emphasized. The
likely importance of neutrophils is demonstrated by detection of the antineutrophil
cytoplasmic antibody (ANCA) in this inflammatory bowel disease. Pyoderma Gangrenosum
(PG) is an idiopathic skin condition and a rare cutaneous manifestation of UC.
ANCA has also been reported in the latter dermatosis.
OBJECTIVES: To invetigate the relationship between clinical features of UC and the appearance of PG and its association with ANCA.
PATIENTS AND METHODS: ANCA was determined in sera from eight patients with PG. Four out of eight patients had pyoderma gangrenosum associated-UC, and in four cases no identified systemic disease was associated.
RESULTS: The search for ANCA yielded negative results in sera from all four patients with pyoderma not associated with systemic disease. Two cases with active and extensive colitis associated with PG and primary sclerosing cholangitis (PSC) were positive for ANCA. Sera from two other patients with both UC and PG had negative test results.
CONCLUSIONS: The presence of ANCA in patients with PG associated with UC and PSC suggests that its association with PSC is responsible for ANCA positivity in this subset of patients.
Key-words: antibodies, antineutrophil cytoplasmic; cholangitis, sclerosing; pyoderma gangrenosum; colitis, ulcerative.
Pyoderma gangrenosum (PG) is classified as a neutrophilic dermatosis. It is characterized by long-term cutaneous ulceration, often triggered by a skin trauma (pathergy).1,2 Its pathogenesis is unknown. Immune system changes have been reported, accompanied by changes in polymorphonuclear leukocyte homeostasis.3,4 Its development during granulocyte colony-stimulating factor therapy,5 as well as its response to immunosuppressive drugs,6 reinforce the relevance of neutrophils and cytokines in the pathogenesis.3,5
PG is regarded as a cutaneous manifestation of several systemic diseases, such as inflammatory bowel disease (IBD), whose prevalence is variable.1,7 PG usually emerges during active bowel disease, although it may be found in the absence of active inflammation, either preceding bowel symptoms or appearing after total colectomy at the site of ileostomy.8-10 Seronegative arthropathy, associated with colonic inflammation, may favor the appearance of PG. There is no agreement regarding the relationship between PG and the extension, duration and severity of the inflammatory bowel disease (IBD).11,12
Rheumatoid arthritis, myeloproliferative disorders, liver diseases, monoclonal gammopathies, Wegener's granulomatosis (WG) and diabetes mellitus6 are also associated with pyoderma, although malignant pyoderma may represent a dermal manifestation of WG. In approximately 50% of cases no associated disease can be identified.4,13,14
Diagnosis is based on the lesion morphology, clinical course, presence of an associated systemic disease and exclusion of possible etiologic agents, since histological findings are nonspecific and no pathognomonic laboratory alterations can be found.2,15
Antineutrophil cytoplasmic antibody (ANCA) has been recently reported in both neutrophilic dermatoses11,16,17 and IBD as a possible serum marker for UC. It is apparently not dependent on inflammatory activity, extension of the bowel disease or on the presence of extraintestinal manifestations.18,19
This antibody was first reported in Wegener's granulomatosis20 and, later, in other systemic vasculitides.21 Using indirect immunofluorescence (IIF), on ethanol-fixed neutrophils, three patterns of fluorescence can be identified: cytoplasmic or c-ANCA, characterized by a densely granular cytoplasmic fluorescence directed against proteinase 3 (PR-3), perinuclear (p-ANCA) that may be related to several antigens, yet typically described as anti-myeloperoxidase (MPO), and the atypical pattern, with fine granular cytoplasm fluorescence becoming more marked around the nucleus, whose antigenic specificity remains to be determined.22,23
Difficulties in characterizing possible factors that favor the development of PG in ulcerative colitis patients, led us to analyze the clinical behavior of UC (gastrointestinal symptom time, extension and activity of colonic inflammation) when associated with PG, the positivity of ANCA in such patients as well as in those with PG without an associated systemic disease.
PATIENTS AND METHODS
Four patients had pyoderma gangrenosum associated-UC, two of whom presented with further extraintestinal involvement (one with primary sclerosing cholangitis (PSC) and the other with PSC and seronegative non-deforming arthropathy) (Table 1). Inflammatory activity of UC was defined according to the Truelove & Witts' criteria at the time of sera collection.24
The PG group without a detected systemic disease comprised four patients (three women), mean age 56 years (36-72) (Table 2). They had lesions on their lower legs and feet. Diagnosis was based on skin biopsy findings with negative cultures for fungi, bacteria and mycobacteria. Polymerase chain reaction (PCR) was not performed. Halogenoderma was excluded.
For pyoderma-associated systemic diseases, the following investigations were analyzed: blood glucose levels, full blood cell count, liver biochemical profile, protein and serum immunoglobulin electrophoresis, rheumatoid factor and chest X-ray. With regard to IBD association, the patients were queried about gastrointestinal symptoms and according to the symptom reported, colonoscopy and/or barium small bowel series were performed (Table 2).
Patient serum samples were analyzed for ANCA using IIF following the standardization proposed at the first workshop on ANCA.27 For antigen-antibody reaction visualization fluoresceine-conjugated goat F(ab')2 anti-human IgG, at a 1.150 dilution was used (Sigma F- 1641, USA), with slide interpretation performed by two independent observers.
Two serum samples, known to be positive for ANCA (cytoplasmic and perinuclear patterns) and a negative one served as controls. Fluorescence at > 1/ 20 dilution was defined as a positive test.
ANCA was detected in two of the four UC patients associated with PG and PSC, both with pancolitis and moderate inflammatory activity (Table 1). Perinuclear fluorescence patterns of 1:80 titer and a nuclear (granulocyte specific antinuclear antibody GS-ANA) titer of 1:160 were found. All sera from PG patients without associated systemic disease were negative for ANCA, and laboratory investigations were unremarkable (Table 2).
The actual relationship between UC and the onset of extraintestinal manifestations is under discussion. The presence of bacterial antigens in the intestinal lumen and their absorption through the damaged colonic mucosa could trigger and perpetuate a local and systemic inflammatory response, arising from both stimulation of the immunological cells and production of pro-inflammatory cytokines. The existence of an antigenic relationship between bacterial antigens and colonic mucosa, biliary tract, skin and/or joints, would render such organs actual "target antigens", which could explain their dysfunctions.12,28
The first publications on PG associated this manifestation almost exclusively with IBD, and such association was later found not to be so frequent.6,7
A further issue for discussion would be whether the onset of pyoderma is dependent on UC extension and active inflammation.9,11,12 Controversial published results may be due to unsuspected colonic mucosa inflammatory activity, according to routine laboratory methods or clinical presentation. Its description in total colectomy patients,8 suggests that colon presence is not the main factor for developing this skin lesion. Among our four patients with UC and pyoderma, three presented with active pancolitis.
PG association with long term UC could define the skin condition as secondary within the chronic inflammatory process. However, most authors report its onset within the first 10 years of inflammatory bowel disease,9,12 like the one found in our cases.
Two of our UC patients with ANCA positive concurrently developed PSC. Rump et al.29 reported a relationship between extraintestinal manifestations and ANCA in UC patients, without specifying what would account for such association. As this antibody has been identified in sera from patients with PSC alone or accompanied by UC,19 a question arises as to whether this hepatobiliary disease is the determining condition for the presence of ANCA in sera from our patients, or whether the association of more than one extraintestinal manifestation favors a greater antigenic response.
Regarding fluorescence, a perinuclear pattern is the most frequent type reported in patients with UC. GS-ANA pattern has not yet been defined, though the absence of fluorescence in lymphocytes upon studying ANCA defines it as directed against neutrophils. Identifying the antigen responsible for this staining pattern could elucidate its meaning.22,28
Upon analyzing the cases of neutrophilic dermatosis positive for ANCA which have been reported in the literature, all were found to be associated with either systemic vasculitides or monoclonal gammopathy, particularly IgA.16,29 Since pyoderma gangrenosum had already been described in association with Wegener's granulomatosis, and since ANCA was defined as a serum marker for that granulomatous vasculitis, Thieme et al.30 investigated ANCA in patients with pyoderma not associated with vasculitis and in those with vasculitis (Wegener's granulomatosis) presenting with that cutaneous manifestation. They did not confirm the relationship between PG and such an antibody.
The same result was found in our study ANCA was shown to be negative in sera from patients with pyoderma not associated with vasculitis. As far as the possible association with IBD in this group of patients is concerned, we chose to investigate only the symptomatic patients or those with laboratory alterations that would suggest the presence of colonic inflammation.
We admit that at least two factors could have influenced the negative result in our series: the small number of patients and the use of a conjugate directed only against the IgG immunoglobulin class. Other authors using conjugates also containing anti-IgA, which is an immunoglobulin frequently responsible for gammopathy associated with PG, would justify the antibody's positivity in such cases.11,16,29
Investigations on a larger number of patients with PG subdivided according to the presence or absence of vasculitis, inflammatory bowel disease, PSC and gammopathy may be able to establish the actual relationship between the skin condition and such clinical entities as well as the role of ANCA in that dermatosis.
We acknowledge the assistance of Prof. Dr. Alice de O. A. Alchorne (Heliópolis Hospital) for referring cases of pyoderma gangrenosum not associated with UC.
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in November, 26th of 2002.
Approved by the Consultive Council and accepted for publication in September, 25th of 2003.
* Work done at the Department of Medicine, Clinical Gastroenterology Section, UNIFESP/EPM.