J Clin Pathol 1994;47:257-262

Neutrophil cytoplasmic antibodies (p-ANCA) inulcerative colitis

PM Ellerbroek, M Oudkerk Pool, B U Ridwan, KM Dolman, B M E von Blomberg,A E G Kr von dem Borne, S GM Meuwissen, R Goldschmeding

Department ofImmunologicalHaematology, CentralLaboratory ofTheNetherlands RedCross BloodTransfusion Service,Amsterdam, TheNetherlandsPM EllerbroekB U RidwanKM DolmanA E G Kr von dem BorneR GoldschmedingDepartment ofGastroenterology ofthe Free UniversityHospital, AmsterdamM Oudkerk PoolB U RidwanB M E von BlombergS GM MeuwissenDepartment ofHaematology,Academic MedicalCenter, AmsterdamA E G Kr von dem BorneDepartment ofPathology, AcademicMedical Center,AmsterdamR GoldschmedingCorrespondence to:Dr R Goldschmeding,Department of Pathology(H2), Academic MedicalCenter, Meibergdreef 9,1 105 AZ Amsterdam, TheNetherlands.Accepted for publication7 October 1993

AbstractAims-To study ulcerative colitis associ-ated neutrophil cytoplasmic antibodies(p-ANCA) in respect of class and sub-class distribution, antigen specificity,and (sub)cellular localisation of the anti-gen(s) to which these antibodies are

directed.Methods-p-ANCA positivity was deter-mined using the standard indirectimmunofluorescence test (IFF). Theimmunoglobulin (Ig) subclass distribu-tion of p-ANCA was investigated usingmonoclonal antibodies directed againstIgGi, IgG2, IgG3, and IgG4. Intracellularantigen localisation studies were per-formed on (fractionated) neutrophilsusing antigen-specific antibodies.Results-In contrast to vasculitis associ-ated ANCA, ulcerative colitis p-ANCAare mainly ofIgGi and IgG3 subclass andlack IgG4. Ulcerative colitis p-ANCAare myeloid specific. IIFT data indicatethat the related antigen(s) seem(s) to belocated not in the cytosol, but in the gran-ules (most likely the azurophil granules)ofthe neutrophil.Conclusions-p-ANCA in ulcerative coli-tis have a different immunoglobulin sub-class distribution than the ANCA ofsystemic necrotising vasculitis andnecrotising and crescentic glomerulo-nephritis. This may point to differencesin immune regulation between these dis-eases. Both cathepsin G and lactoferrinare recognised by a subpopulation ofulcerative colitis p-ANCA. In our series,eight out of 36 (22%) of ulcerative colitisassociated p-ANCA react with lactoferrinand seven (19-5%) other sera with cathep-sin G. None of them recognised bothantigens. The main target antigen(s) ofulcerative colitis p-ANCA still remain(s)to be identified.

( Clin Pathol 1994;47:257-262)

Anti-neutrophil cytoplasmic antibodies(ANCA) have proved their value in the diag-nosis of vasculitic disorders, such as

Wegener's granulomatosis, systemic necrotis-ing vasculitis, and necrotising crescenticglomerulonephritis.' In these diseases theANCA titre correlates with disease activity.Exacerbations are preceded by a rise in theANCA titre in the patient's serum, so theseantibodies may have a pathogenetic role.1-3

The related antigens have been identified asenzymes located in the granules of neu-trophils. C-ANCA in Wegener's granulo-matosis are directed against proteinase 3.p-ANCA in systemic necrotising vasculitisand necrotising crescentic glomerulonephritisare mainly directed against myeloperoxidaseand rarely against neutrophil elastase.49

In 1983 Nielsen et al described (peri-)nuclear staining of neutrophils fixed inethanol in the sera of patients with ulcerativecolitis, using a fixed cell indirect immunofluo-rescence technique (IIFT). This pattern wassimilar to that of granulocyte specific antinu-clear antibodies (GS-ANA) found in the seraof patients with rheumatoid arthritis. It hasbeen reported in several studies that theseantibodies are present in the sera of 25% ofpatients with ulcerative colitis and only in 3%of those with Crohn's disease.'01' Saxon et alreported similar perinuclear staining of neu-trophils, now called p-ANCA, in the serafrom patients with inflammatory bowel dis-ease. In their study 68% (n = 25) of the ulcer-ative colitis sera and 12% (n = 25) of theCrohn's disease sera reacted positively in theIIFf. 12 We recently screened 317 sera frompatients with inflammatory bowel disease and49 control sera using the IIT; 79% of theulcerative colitis sera, 13% of the Crohn's dis-ease sera, and 9% of the control sera showed apositive reaction with neutrophilic granulo-cytes. p-ANCA titres were not correlated inulcerative colitis or in Crohn's disease withdisease activity, duration of illness, localisa-tion, extent of disease, previous bowel opera-tions or medical treatment. The clinicalimportance of p-ANCA positive and negativesubsets in both groups of patients thusremains unexplained.'3 To obtain a betterinsight into the background and importanceof this autoimmune phenomenon in ulcerativecolitis, we therefore investigated the size, classand the subclass distribution of ulcerative col-itis related p-ANCA immunoglobulins as wellas the subcellular localisation of the antigen(s)to which these antibodies are directed.

MethodsSera from 36 patients with ulcerative colitis,positive for IgG p-ANCA staining in the IIFTon ethanol-fixed granulocytes, were selectedfor further study. The presence of antinuclearantibodies in the sera was excluded in a rou-tine assay using rat liver as a substrate.Ulcerative colitis was diagnosed on the basisof clinical, radiological, histological, and

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endoscopic evidence.'4 Sera containing anti-bodies against myeloperoxidase, proteinase 3,elastase, lactoferrin, and cathepsin G, asdetected by capture ELISA, served as controlsin all the assays used in this study. Sera nega-tive in the IIFT were obtained from patientswith Crohn's disease and from healthy blooddonors to serve as controls.

BLOOD CELLS AND LEUKAEMIA CELL LINESGranulocytes and lymphocytes were obtainedby Ficoll sedimentation of EDTA anticoagu-lated buffy coats of regular blood donationsfrom healthy donors. Leucocytes were alsoisolated from blood of a patient with chronicmyeloid leukaemia (CML). Lymphocyteswere aspirated from the interface and washedin 0-2% bovine serum albumin-phosphatebuffered saline (BSA-PBS). Erythrocytes werelysed by ammonium chloride and the remain-ing granulocyte pellet was washed three timeswith PBS. Thrombocytes were isolated bycentrifuging EDTA anticoagulated blood at400 x g, followed by centrifugation of theplatelet rich plasma at 1500 x g. The pelletedplatelets were suspended in EDTA-PBSbuffer. Monocytes were isolated by successiveisopycnic centrifugation and counterflow elu-tion, as described by Roos et al.15 The histio-cytic cell line U937 was cultured in RPMI1640 medium (Gibco, Paisley, Scotland).

PREPARATION OF CYTOPLASTS ANDKARYOPLASTSGranulocytes from a healthy donor were frac-tionated by discontinuous Ficoll gradient cen-trifugation with cytochalasin B (at 37°C).After washing in PBS the cytoplasts (contain-ing two thirds of the cytosol surrounded byplasma membrane) and karyoplasts (the gran-ules, nuclei and one third of the cytosol sur-rounded by plasma membrane) were eithercytospinned and fixed in ethanol for IIFT, orcoated as a monolayer to microtitre plates inHanks's buffered salt solution and fixed with100% methanol for enzyme linked immuno-sorbent assay (ELISA).'6

PREPARATION OF NEUTROPHIL GRANULEPROTEIN EXTRACTIsolated neutrophils were disrupted by cavita-tion in a nitrogen bomb.'7 Nuclei and unbro-ken cells were pelleted by centrifugation at500 x g for 10 minutes (at 4°C). The super-natant fluid was centrifuged at 35 000 x g for20 minutes (at 4°C). The pelleted mixedgranules were resuspended in PBS-0-1% (v/v)Triton X-100 and sonicated at 45 kHz onmelting ice for three periods of 10 secondseach. Thereafter, soluble and insoluble mater-ial were separated by centrifugation at220 000 x g for 1 hour (at 4°C). The super-natant "extract" was dialysed against PBS.17

IIFTThe standard IIFT was performed accordingto Wiik's method.'8 Isolated human bloodcells were either cytospinned on slides(Menzel Glaser, Omnilabo International,Breda, The Netherlands) or smeared on

eight-well Nutacon slides (Nutacon,Schiphol, The Netherlands) and fixed in 96%ethanol for 15 minutes (at 4°C). Thereafter,the slides were incubated with serum diluted1 in 16 in 4% BSA-PBS for 1 hour, followedby washing in PBS. Bound antibodies weredetected by fluorescein isothiocyanate(FITC)-labelled F(ab')2 fragments of rabbitanti-human IgG. The slides were examinedby fluorescence microscopy. In some experi-ments the smears were fixed with phosphatebuffered formalin and acetone solution,pH 6-8, containing 9-25% (v/v) formalin and45% (v/v) acetone for 2 seconds at 40C. Theimmunoglobulin subclass distribution ofp-ANCA in ulcerative colitis was investigatedusing monoclonal antibodies directed againstIgGl, IgG2, IgG3, and IgG4, followed byFITC-conjugated goat anti-mouse immuno-globulin. For the detection of IgM and IgAbinding, FITC-labelled F(ab')2 fragments ofrabbit anti-human IgM or IgA were used. Theoptimal dilutions were determined by titrationon positive and negative control sera.To examine the reactivity of sera with the

plasma membrane, granulocytes were fixed insuspension with 1% paraformaldehyde for5 minutes (at 4°C). Bound antibodies weredetected by fluorescein-conjugated F(ab')2fragments of sheep anti-human immuno-globulin and measured by fluorescence flowcytometry. 9

CAPTURE ELISAThis assay is described in detail in reference 5.In brief, goat anti-mouse IgG antibodies(Jackson Immuno Research Laboratories Inc,Avondale, Philadelphia, USA) were coated onmicrotitre plates (Maxisorp, Nunc). Afterincubation with monoclonal antibodies andwashing, the plates were incubated with gran-ule extract to allow the monoclonal antibodiesto capture their respective antigens from aneutrophil granule extract. After another washpatient serum was added. Bound patient anti-bodies were detected by affinity purified alka-line phosphatase conjugated goat anti-humanIgG.5

Ficoll, protein A-sepharose CL-4B, andsephacryl S-400 were obtained fromPharmacia Fine Chemicals (Uppsala,Sweden). Paraformaldehyde, formalin, andmethanol were purchased from Merck(Darmstadt, Germany). Acetone was fromOPG Farma (Utrecht, The Netherlands).Ethanol was from Brocacef (Maarssen, TheNetherlands), and EDTA from AldrichChemie (Brussels, Belgium). Cytochalasin B,Triton X-100, and Sigma 104 alkaline phos-phatase substrate were from Sigma (St Louis,Missouri, USA). Bovine serum albumin wasobtained from Boseral (Boxtel, TheNetherlands). ACA 44 was from IBF(Villeneuve la Garenne, France).

ATISERAFITC-labelled F(ab')2 fragments of rabbitanti-human IgG were obtained fromDakopatts D/A (Denmark). Alkaline phos-phatase labelled goat anti-human IgG/goat

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Neutrophil cytoplasmic antibodies (p-ANCA) in ulcerative colitis

Figure 1 IgG, IgM, andIgA class titres of ulcerativecolitis related p-ANCAs,anti-proteinase 3 ANCAs,and anti-myeloperoxidaseANCAs.

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anti-mouse IgG and unlabelled goat anti-mouse IgG were obtained from JacksonImmuno Research Laboratories Inc. FITC-coupled F(ab')2 fragments of sheep anti-human IgG (S26H17F), FITC-coupled goatanti-mouse IgG, goat anti-human IgG, rabbitanti-human IgM, peroxidase bound rabbitanti-human IgM, FITC-coupled rabbit anti-human IgA (KH26H14), and FITC-coupledrabbit anti-human IgM (K26H15) wereproduced at the Central Laboratory of theNetherlands Red Cross Blood TransfusionService (Amsterdam) as were the monoclonalantibodies anti-human IgGl (clone 161.2M),anti-human IgG2 (clone 162.1M), anti-humanIgG3 (clone 163. 1M), anti-human IgG4 (clone164.4M), and 12.8 (IgGl anti-proteinase 3),4.15 (IgGI anti-myeloperoxidase), 14.15 (IgGIanti-neutrophil lactoferrin), and AME-1(IgGl anti-glycophorin A). AHN-11 (IgGlanti-cathepsin G) was a gift from Dr KMSkubitz (University of Minnesota MedicalSchool, Minneapolis, USA). NP-57 (IgGlanti-neutrophil elastase) was a gift from DrDY Mason (John Radcliffe Hospital, Oxford,England).

ResultsIgG CLASS AND SUBCLASS DISTRIBUTION OFULCERATIVE COLITIS ASSOCIATED p-ANCAIn most ulcerative colitis p-ANCA sera IgGclass antibodies only were detected.Additional IgM p-ANCA binding was foundin three out of 36 sera and IgA p-ANCA in 10out of 36 sera. The occurrence of IgM andIgA class ANCA was also tested in 20 controlsera, either positive for IgG class ANCAdirected against proteinase 3 (n = 10) or IgGp-ANCA against myeloperoxidase (n = 10)(fig 1). Anti-proteinase 3, as well as anti-myeloperoxidase p-ANCA, were also mainlyIgG class, but in six out of 10 sera with IgGantibodies against myeloperoxidase there wasa strong IgM class p-ANCA as well. Thesesera did not contain rheumatoid factor, asdetermined by the latex fixation test.

Ulcerative colitis p-ANCA IgG was mainly

of IgGl and IgG3 subclasses. No IgG4 wasdetected (fig 2A). In contrast, vasculitis-associated anti-proteinase 3 ANCA and anti-myeloperoxidase ANCA IgG containedrelatively high concentrations of specific IgG4(fig 2B and C).

FIXATION STUDIESTo study the effects of different fixation pro-cedures on the staining patterns obtained inthe IIFT, 20 ulcerative colitis p-ANCA serawere tested both on cytospins of granulocytestreated with ethanol (standard IIFIT) and onslides treated with phosphate buffered forma-lin-acetone solution. On formalin-acetonefixed cells, three out of 20 ulcerative colitisp-ANCA sera were negative; three other seraalso showed vague cytoplasmic staining. Theremaining (14/20) sera showed a granularcytoplasmic staining pattern, although weakerin intensity than that on ethanol fixed cells(not shown).

CELL SPECIFICITYAll ulcerative colitis p-ANCA positive serawere also assayed for reactivity with cytospinslides of thrombocytes, lymphocytes, andeosinophilic granulocytes. None of these cellswas stained by ulcerative colitis p-ANCA sera.When tested on monocytes, however, 21 serashowed p-ANCA reactivity with 10-40% ofthe monocytes. Thirteen p-ANCA positivesera also reacted positively with somepromonocytic U937 cell line cells. Thusulcerative colitis associated p-ANCA aremyeloid specific and a 100% reaction is foundonly with neutrophilic granulocytes (table).

REACTIVITY WITH ISOLATED MYELOIDGRANULE PROTEINSIn contrast to control c-ANCA and p-ANCApositive sera from patients with Wegener'sgranulomatosis and other vasculitides, ulcera-tive colitis p-ANCA sera did not react withcaptured proteinase 3, myeloperoxidase, orelastase. A few of the ulcerative colitis sera didrecognise lactoferrin or cathepsin G. Eight of36 sera bound to lactoferrin. Seven other sera

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IgG subclass ulcerative colitis ANCA(n = 36)

IgGl IgG2 IgG3

Reactivity ofp-ANCA positive ulcerative colitis sera withdifferent cell types in IIFT

Positive sera Percentage ofCell type (total n = 36) cells stained

Neutrophils 36 100Eosinophils 0 0Monocytes 21 10-40Lymphocytes 0 0Thrombocytes 0 0U937 13 10-40

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bound to cathepsin G in a capture ELISAusing granule extract from chronic myeloidleukaemia cells (fig 3). Three of these sevensera showed only marginally increased binding.

SUBCELLULAR LOCALISATION OF p-ANCAASSOCIATED ANTIGEN(S)Using immunofluorescence on paraformalde-hyde-fixed neutrophils in suspension (IIFT),none of the ulcerative colitis p-ANCA serareacted positively with the granulocyte plasmamembrane. Using IIFT on cytospins as wellas a solid phase ELISA, none of 10 ulcerativecolitis p-ANCA sera tested reacted with thecytoplasts of neutrophils, but all 10 serareacted positively with the karyoplast fractionof the same cells. Therefore, the antigensrecognised by ulcerative colitis p-ANCA donot seem to be located in the cytosol orplasma membrane, but in cellular structureswith higher sedimentation rates-the gran-ules, for example.

In additional experiments using solid-phaseELISAs 11 out of 36 ulcerative colitis p-ANCA sera reacted with a crude preparationof (N2-cavitated) neutrophil granule proteinextract from which the nuclei and cytosol hadbeen removed by differential centrifugation.The same 11 sera reacted positively with acomparable preparation extracted from CMLcells. Five other sera reacted positively onlywith the CML preparation but not with thatfrom mature neutrophils. Reactivity with thevarious preparations in these ELISAs was notrelated to the titre of the sera in the IIFT.Western blotting and radioimmunoprecipita-tion did not identify bands specifically recog-nised by ulcerative colitis p-ANCA sera (datanot shown).

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Figure 2 (A) IgG subclass distribution ofp-ANCA; (B) anti-proteinase 3 ANCAs; (C)anti-myeloperoxidase ANCAs as detected by IIFT. To enable comparison of results withsera with different (total) IgGANCA titres for each serum, the IgG subdass titre isexpressed relative to that obtained with FITC coupled goat anti-human IgG (total).

Figure 3 Capture ELISA for detection of antibodiesagainst lactoferrin and cathepsin G. Thirty six p-ANCApositive ulcerative colitis sera were tested. None of the serarecognised both antigens. Lines indicate means ± 2 SD of10 normal control sera included in this experiment. Normalrange is representative as evidenced by separate experimentsincluding larger numbers of controls (not shown).

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Neutrophil cywplasmic antibodies (p-ANCA) in ulcerative colitis

DiscussionWe investigated the nature of the neutrophilreactive immunoglobulin(s) in ulcerative colitissera that produce p-ANCA staining in theIIFT on ethanol fixed neutrophil slides. p-ANCA in ulcerative colitis sera, like manyother autoantibody sera, are mainly IgG classantibodies of the IgGl and the IgG3 subclassand lack IgG4 antibodies. In this respect,however, they differ from the vasculitis associ-ated ANCAs which have relatively high IgG4titres. This suggests that there may be differ-ences in the regulation of the autoimmuneresponse in ulcerative colitis compared withthe vasculitides.2223

Like systemic necrotising vasculitis andnecrotising and crescentic glomerulonephritisassociated ANCAs, p-ANCA in ulcerativecolitis seem to be myeloid specific.' In theIIFT ulcerative colitis p-ANCA reacted withboth neutrophils and monocytes, althoughgenerally only a minority of monocytesbecame positive (table). This differential reac-tivity with monocyte(-subpopulation)s mightbe related to differences in antigen contentsbetween immature and mature monocytes.24The fact that, in contrast to our observations,Duerr et al found no reactivity of ulcerativecolitis sera in a fixed monocyte ELISA mightbe explained by lower sensitivity of this testfor identification of antibodies reacting withonly subpopulations of these cells.25 It is possi-ble that the ulcerative colitis associatedGS-ANA, originally described by Nielsen inthe early eighties are not nuclear but in factperinuclear staining patterns, that are nowcalled p-ANCA.'01' This p-ANCA pattern,however, is not a reliable indication of thesubcellular location of the antigen(s) involved.It may be artefactually generated by the fixa-tion process of neutrophils. The ulcerativecolitis p-ANCA staining pattern in the LIFT'depended on the fixation procedure, as hasbeen described earlier for vasculitis associatedp-ANCA directed against myeloperoxidaseand elastase.26 For ulcerative colitis p-ANCA,we found a perinuclear staining pattern onethanol fixed cytospins or smears; after forma-lin-acetone fixation the pattern was cytoplas-mic. This suggests that, similar to the targetantigens of vasculitis-related ANCA, theulcerative colitis p-ANCA antigen(s) may alsobe located in the granules, rather than in thenucleus of neutrophils.27 The results of cellline studies, cytoplast/karyoplast separationand granule extract analysis would be compat-ible with localisation of the target antigen(s)of ulcerative colitis p-ANCA in the azuro-philic granules of neutrophils, like the targetantigens of systemic necrotising vasculitis andnecrotising and crescentic glomerulonephritisANCA.57 Only a minority of p-ANCA posi-tive ulcerative colitis sera, however, gave apositive result in solid phase ELISAs on neu-trophil and CML granule extracts. No specificbands could be identified by western blottingor radioimmunoprecipitation. In our routinecapture ELISA for detection of vasculitis andglomerulonephritis related ANCA againstproteinase 3, myeloperoxidase, and elastase,

all ulcerative colitis p-ANCA sera were nega-tive. In addition, in a solid phase ELISA ondirectly coated antigens (purified proteinase3, myeloperoxidase, elastase) no clear positivereactions were obtained. Ulcerative colitis p-ANCA, therefore, do not seem to be directedagainst the same antigens as systemic necro-tising vasculitis and necrotising and crescenticglomerulonephritis ANCA.Thompson and Lee reported the detection

of antibodies against lactoferrin in a patientwith Wegener's granulomatosis but these anti-bodies do not seem to be common in this dis-ease.28 A recent study suggests that lactoferrinantibodies may be an important target antigenof p-ANCA in patients with ulcerative colitiswho also have primary sclerosing cholangitisas IgG lactoferrin antibodies were found in50% of the sera from such patients.29Halbwachs-Mecarelli et al reported p-ANCAactivity against cathepsin G in a substantialproportion ofp-ANCA positive sera in a smallgroup of ulcerative colitis patients.30

Although we found smaller percentages ofpositive sera, the results of our study confirmboth antigen specificities. In eight out of 36 p-ANCA positive ulcerative colitis sera wedetected antibodies against lactoferrin; sevenother sera contained antibodies againstcathepsin G. The remaining 21 ulcerative col-itis p-ANCA sera failed to show such reactiv-ity. Efforts to characterise other antigensinvolved in ulcerative colitis p-ANCA reactiv-ity, by Percoll density gradient centrifugationand immunoprecipitation, which have beensuccessfully used in the identification of thetarget antigens of systemic necrotising vasculi-tis and necrotising and crescentic glomeru-lonephritis ANCA,5 have thus far not revealedtheir identity. Theoretically, this might alsorelate to the possibility of p-ANCA stainingbeing produced by adherence of "sticky"immune complexes or aggregates rather thanby antigen specific immunoglobulin binding.Therefore, size distribution of ulcerative coli-tis p-ANCA reactivity was studied usingulcerative colitis p-ANCA serum lacking IgMor IgA class p-ANCA after isokinetic sucrosegradient centrifugation.? p-ANCA reactivitywas found within the monomeric IgG peak(not shown). In addition, gel filtration at acidpH (performed by Wiik et al to dissociateimmune complexes in GS-ANA sera frompatients with rheumatoid arthritis20) did notreveal p-ANCA reactivity in immune com-plexes (not shown). Together with the gener-ally weak and variable reactivity of theulcerative colitis p-ANCA sera in the variousassays used, this might suggest that ulcerativecolitis p-ANCA are monomeric but low affinityantibodies, which may be generated in thecourse of chronic inflammation. Becauseproduction of IgG4 antibodies is related toprolonged antigen specific T cell dependentimmune stimulation, the lack of IgG4 inulcerative colitis p-ANCA would also seemcompatible with a relatively low affinity ofthese antibodies. Our own observation thatp-ANCA positivity does not correlate withactivity, extent, or duration of ulcerative colitis

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may seem to argue against chronic inflamma-tion as a factor in the generation of ulcerativecolitis p-ANCA.'3 It should be realised, how-ever, that ulcerative colitis is by definition adisease of chronic inflammation and that theformation of p-ANCA in only a subgroup ofulcerative colitis patients may depend on dif-ferences in genetic background.32

In conclusion, unlike the ANCA of sys-temic necrotising vasculitis and necrotisingand crescentic glomerulonephritis, which arepredominantly of the IgGl and IgG4 sub-classes, p-ANCA in ulcerative colitis aremainly of IgGl and IgG3 subclasses and lackIgG4, which may point to differences inimmune regulation between these groups ofpatients. We have clearly shown that bothcathepsin G and lactoferrin are recognised by aminority of ulcerative colitis p-ANCA sera,but none of them are recognised both anti-gens. Finally, we have excluded the possibilitythat these antibodies are artefacts related tocirculating immune complexes in thesepatients.

We thank Professor AS Pena for helpful comments.

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2 Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neu-trophil cytoplasmic autoantibodies induce neutrophils todegranulate and produce oxygen radicals in vitro. 1ProcNatlAcad Sci USA 1990;87:4115-19.

3 Kallenberg CGM, Cohen Tervaert JW, van der Woude El,Goldschmeding R, von dem Borne AEG, Weening JJ.Viewpoint autoimmunity to lysosomal enzymes: newclues to vasculitis and glomerulonephritis? ImmunologyToday 1991;61:61-4.

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13 Oudkerk Pool M, Ellerbroek PM, Ridwan BU, et al.Serum antineutrophil cytoplasmic autoantibodies ininflammatory bowel disease are mainly associated withulcerative colitis. A correlation study between perinu-clear antineutrophil cytoplasmic autoantibodies andclinical parameters, medical, and surgical treatment. Gut1993;34:46-50.

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