Clin Exp Immunol 1994; 95:49-55

Antineutrophil cytoplasm antibodies (ANCA) of IgA isotype inadult Henoch-Schonlein purpura

N. RONDA. V. L. M. ESNAULT. L. LAYWARD*. V. SEPE. A. ALLEN*. J. FEHHALLY* & C. M.LOCKWOOD Department of Medicine. School of Clinical Medicine. University of Cambridge. Cambridge and

* Department of Nephrology. Leicester General Hospital. Leieester. UK

(Accepted for publieation 21 September 1993)

SUMMARYANCA are associated with certain forms of systemic vasculitis, and have been reported previously tobe of Ihe IgG and IgM isotype. We examined ihc possible association between IgA ANCA and theIgA-relaled diseases Henoch-Schonlein purpura (HSP) and IgA nephropaihy (IgAN). IgA and IgGANCA were detected by isotype-specifie solid-phase assays with a erude neutrophil extract, and theirpresence was confirmed by antigen-specific fluid-phase competitive inhibition tests and by indirectinimunofiuorescence. The possible interference by IgA rheumatoid faeior was excluded. IgA ANCAwere detected in sera from 11/I4 HSP patients (79%). from 1/30 IgAN paLicnts 0"A>), from 1/40patients with vasculitldes classically associated with IgG ANCA (2-5%), and in none or60 sera fromhealthy blood donors. IgG ANCA were present with IgA ANCA in three patients with HSP. Onlyone HSP serum had anti-myeloperoxidase (MPO) activity by both IgA and IgG isotypc-specificELISA, and none was positive for proteinase 3 (PR3). Western blot analysis performed withneutrophil extract showed that the four strongest IgA ANCA-positive HSP sera reacted with a 51-kDprotein; Western blot performed on cellular fractions showed that this protein is primarilymembrane-assoeiated, and different from fibronectin. Our study suggests that adult HSP is eloselyassociated with cireulating IgA ANCA. which may be directed against a different autoantigen thanthat recognized by IgG ANCA.

Keywords antineutrophil eytoplasmic antibodies autoimmunity IgAHenoeh Schontein purpura systemie vasculitis

INTRODUCTIONThe vasculitis syndromes comprise a spectrum of diseases as yetstill defined by clinical and histological criteria [1.2]. Theassociation of ANCA with Wegener's granulomatosis [.1],microscopic polyarterilis [4] and renal limited vasculitides [5]has indicated that autoimmune mechanisms may underlie thedevelopment of these diseases, and increasing evidence points totheir importance in pathogenesis 16]. Studies so far indicate thatANCA are predominantly ofthe IgG isotype. However. ANCAof IgM isotype (without IgG) have been found in patients with apulmonary/renal, anti-glomerular basement membrane (GBM)negative syndrome [7], as well as in association with IgG ANCAin patients presenting with pulmonary haemorrhage [8],

Henoeh Schonlein purpura (HSP) is a systemic vasculitis.more eommon in children, but well documented in adults [9],whieh is associated with IgA abnormalities. These include raisedcirculating IgA levels [10.11]. the presence in serum of IgA-containing immune complexes [10.12.1.3] and IgA deposits in

Correspondence: Dr Nieolelta Ronda, Istiiulo di Clinica Mediea eNefrologia. tJniversita degli Studi, v. Gramsci, 14.4:illOO Parma. Italy.

skin [14.15]. as well as renal mesangium [14.16]. The same renalimmunopathologie and serologic IgA abnormalities are lypi-cally found in igA nephropathy (IgAN). and the two diseasescan be distinguished only by the extrarenal vasculilic involve-ment in HSP [15.17.18].

IgA ANCA have been reported lo occur in HSP [19]. but asyel this finding has not been eonfirmed by others [20], and il hasbeen pointed oul thai patienl IgA fibronectin complexes mayaccount, at least in part, for some IgA ANCA activity [21]. Weexamined the possible association of IgA ANCA wiih HSP andIgAN. excluding IgA rheumatoid factor interference, andinvestigated autoantibody specificity by means of antigen-speeifie solid-phase immunoassays and Western blotting.

PATIENTS AND METHODSPatients

HSP (n= 14. mean age 45 years, age range 17-76 years, 12male and two female). Palienls included in this group fulfilledthe criteria of the American Rheumatism Association. Renalbiopsy was performed in 12 patients and showed mesangial

49

50 .^ Ronda et al.

deposition of IgA in all cases. Sera were stored from all patientsat presentation, or at times of clinical relapse. None of thepatients was receiving treatment al the time serum was obtained.

IgAN (/I = 30, mean age 36 years, age range 16-60. 24 maleand six female). These patients had impairmcnl of renalfunction (or haematuria/proteinuHa) and diffuse mesangial IgAdeposition, without extrarenal involvement. Sera from all IgANpatients were taken at onset of the disease, and in .six patientsduring remission, as well as during episodes of maeroseopiehaematuria.

!gG ANCA-positive systemic lasciditis (/! 40). Wegener'sgranulomatosis {/( = 9) or mieroscopic polyarteritis (/7 = 31).

Healthy donors (H = 60) . These were studied as negativecontrols (age range 17-64 years, 38 male and 22 female).

ANCA solid-phase immunoa.ssaySereening on the crude neutrophil extraet for IgG ANCA wasperformed as described [4]. Detection of IgA ANCA was byELISA using the same neutrophil preparation, blocking with2"A> bovine serum albumin (BSA); gelatin was used as a blockingagent in a subsequent ELISA performed with the sera whichshowed high reactivity, loexcludenon-specilic binding of IgA toBSA. For this purpose, controls also included antigen-free wellsblocked with BSA or gelatin and incubated with patient andcontrol sera. Serum dilutions were calculated for each samplefrom the total IgA concentrations in order to obtain a linal IgAconcentration of 20 mg/dl in both patient and eontrol sera.Binding was deteeted by adding alkaline phosphatase-eonju-gated polyclona! goat anti-human IgA (Sigma, A9669) diluted1:500. and alkaline phosphatase substrate bulTer. After 40 minabsorbance was measured at 402 nm. Alt volumes were 100 ml/well and all incubations were for I h at 37 C, with triple washesof PBS containing 0 1% Tween 20 (PBS-T) between stages.ELISA posilivity was defined as absorbance > 2 s.d. over themean of values obtained from :t panel of normal sera. IgAANCA-positive and normal control sera were also tested in amodified ANCA solid-phase immunoassay (SPIA), usingmouse anti-human IgA 1 and IgA2 MoAbs (Unipath, M120 i Iand M270ID) and a rabbit anti-mouse peroxidase-eonjugatedantibody (Dako, P260) as revealing system. In this ease boundIgA was revealed with orthophenylenediamine nnd H;0; incitric acid bufler, and reaction blocked after 20 min with 2 Msulphuric acid. Absorbance was read at 492 nm.

Antigen-speeifie assays were performed: igGanti-myeloper-oxidase (MPO) as deseribed [22] and IgG anti-protcinase 3(PR3) using a commercially available kit (Bio-Carb, Sweden)according to the manufacturer's instructions. These assays wereadapted to deteci IgA anti-MPO and IgA anti-PR3 by using thesame detection system as for the total IgA ANCA SPIA,

Inhibition studiesThe specilicity of IgA ANCA binding in the ANCA SPIA wastested by means of a fluid-phase inhibition test [4]. Aliquots ofsera were retested in the ANCA SPIA after incubation for I h at37 C with respectively PBS, haemoglobin imd neutrophilextract at proteineoncentrations ranging from 0 005 to 4 mg/ml.The test was considered to be positive when the reduction inANCA binding was greater than 20"/ii following incubation withneutrophil extract, but not with the irrelevant protein, and whena dose-response related to the neutrophil extract coneentrationwas observed.

Indirect immunojluorescenceIndirect immunofluoreseence (IIF) was performed on ethanol-fixed human neutrophils, according to the method previouslydescribed [4], modified for detection of IgA antibodies by usingan anti-IgA lluorescein-eonjugatcd antibody (Dako, F204)diluted 1:5 in PBS. Serum dilutions used were I; 2, 1:4 and 1:8for each patient and control sample. The fluorescence wasassessed independently by three investigators.

IgA and IgG rheumatoidfacior ELISAA specific ELISA kit for tbe detection oflgA rheumatoid factor(RF) was used on all IgA ANCA-positive sera according to themanufacturer's instructions (SKI.ISA; Walker LaboratoriesLtd., Ely, LIK). The standard curve of IgA RF activity wasobtained using IgA RF-positive serum, provided by the manu-facturer, from a patient with rheumatoid arthritis (fulHIling thecriteria ofthe American Rheumatism and Arthritis Council forRheumatoid Arthritis); the serum had been previously cali-brated against the International Standard of IgA RF in order todeteet from 3 0 to 600 IgA RF U/ml. The presence of IgG anti-IgA RE was tested by ELISA on igA-coated plates.

Two HSP patients' sera and a serum positive for IgA RFwere chromatographed on a Sepiiarose column coupled withProtein A at dilutions of 1:10. 1:20 and I ;40, The loaded andthe effluent fractions were tested for IgA ANCA by ELISA asdescribed.

Criteria for IgA ANCA positivilyPositivity was defined by the association of all the followingcriteria: (i) positive IgA ELISA and antigcn-spccilie dose-dependent fluid-phase inhibition; (ii) positive IgA IIF; and (iii)absence of IgA RK activity.

Western blot for Ig.4 ANCANcutrophils were isolated on a Hypaque gradient [4], resus-pended in PBS containing 0? niM MgCl:. i)*-) niM CaCti andprotease inhibitor (phenylmethylsulfonyltluoride I mM) andincubated at 37 C for 30 min with I mg/ml phorbol myristateaeetate (PMA) to induee the release of secondary granules [23|.After centrifugation the supernatant was removed and stored at 80 C until use in Western biol. nnd the cells washed threetimes with PBS. The cells were then incubated in PBS with 1 "-Triton XlOO. phenylmethylsulfonylfluoride 1 niM and EDTA2 mM at 4 C for 40 min. Following eentrifugation at ,S00 g for5 min at 4 C, the supernatant was frozen at - 8 0 C until use inWestern blot, SDS-PAGE was performed with: (i) the storedsupernatant after PMA stimulation; and (ii) the neutrophilextract in reducing or non-reducing buffer. Western blot wasperformed according to the method described by Laemmli [24].IgA ANCA-positive and control sera were diluted 1:20 in Tris-bulTered saline containing 1",. Tween 20 and 2"',) dried skimmedmilk (TBSTM) and incubated with the nitrocellulose strips for 3h at room temperature. The strips were then washed withTBSTM and incubated for 1 h with alkaline phosphatase-conjugated anti-human IgA (Sigma, A9669), diluted 1:1000 inTBSTM. Binding was detected by adding alkaline phosphatasesubstrate.

In order to study the antigen localization in the neutrophils.cellular fractions were prepared from a total extract, i.e. w ithoutPMA stimulation. Neutrophils were isolated on a Hypaquegradient, washed twiee in PBS and ineubated in PBS containing

IgA ANCA in HSP 51Table I. Orctirrence of IgA and IgG ANCA in Hcnoch Schonleinpurptir;i(KSP). IgA nephropathy (IgAN). IgG ANC'A-posilive vasculi-

tis (SV) patients and conlrols

HSP (%) IgAN (%) SV (%) Conlrols (%)

IgA ANCA

IgG ANCA

11/14(79)3/14(21)

1/30 (3)0/30 (0)

1/40(2-5)40/40 M (HI)

0/60 (0)0/60 (0)

T'/.i Triion XIOO. phenyimethylsulfonylfluoride 1 mM andEDTA 2 mM at 4 C for 40 min. Cellular extract was thencentriftigcd at lOOOOj? for i 5 min at 4 C. The pellet was passedseveral times through a 22 G needle and stored tintil use. Thesupernatant, eontaining solubilized membranes and cytosoliematerial, was divided into two aliquots. One aliquot was storeduntil use and the other was centrifuged at I05000j?ror40minat4 C. Both the pellet and Ihe supernatant obtained with this lasteentrifugation were saved for use. ihe pellet being resuspendedin a volume equivalent to the supernatant. SDS-PAGE wasperformed in reducing conditions with: (i) pellet and super-natant after centrifugation at lOOflO^ (containing respectivelynuclei + coarse fragments of membrane and solubilized mem-branes + eytosolic material); (ii) pellet and supernatant aftercentrifugation al 105 OOOjf (representing respectively solubilizdmembrane-enriched material and soluble cytoplasm). AfterWestern blot. IgA reactivity against each fraction was testedusing an IgA ANCA-positive serum (whieh had previously beenshown to react against the 51-kD protein) and a eontrol serum.

In order to investigate whether IgA-fibronectin interactionscould be responsible for the 5i-kD band of reactivity observedin the Western blot of" HSP IgA, Weslern blot experiments witha total neutrophil extract and the membrane-rich fraction werealso perfonned to test the reactivity of three commerciallyavailable anti-libronectin antibodies (rabbit polyclonal anti-human tibroneclin, mouse monoclonal anti-human fibronectinand mouse monoclonal anti-cellular fibronectin: Sigma. St.Louis. MO). Two IgA ANCA-positive HSP sera were used aspositive controls. Revealing aniibodies were alkaline phospha-tase-conjugated anti-rabbil IgG, anti-mouse IgG. anti-mouseIgM and anti-human IgA.

RESULTSigA ANCA were deteeted in 11/14 (79%) of HSP patients, in 1/M) (3%) of IgAN. and in 1/40 (25%) systemic vasculitis (SV)patienls. None of the sera from the healthy controls was foundlo have IgA ANCA activity (Table 1). IgG ANCA was found iniissocialion wiih IgA ANCA in three of the HSP patients, bulnol in any of the patients with IgAN, nor in healthy controls.

ANCA SPIA with inhibition studyThe resull of the IgA ANCA SPIA Is shown in Fig. I. High totalIgA levels are a common finding in HSP and IgAN patients; wetherefore used for eaeh sample a different serum dilution (from1:10 to 1;33) to obtain 20 mg/dl of total IgA to exclude thepossibility of non-specific binding: this theoretical possibilitywas also excluded by testing each serum on bolh antigen-freewells bloeked with BSA and antigen-free wells blocked withgelatin on the same microlltre plate. None of the positive sera

800 r

600

400

200

Controls HSP IgAN/I = 30)

SV

Fig. 1. IgA ANCA ELISA resulls rehiive to palicnts with HcnochSchonlein purpura (HSP). IgA nephropaihy (IgAN), lg(J ANCA-positlvc sysictitic viisculitis (SV). and conlrols. On the f-axis is theoptical density (OD). Bars show tneanis.d. for each population. Thedotted line represents the upper normai value (mean + 2 s.d. of valuesfrom controls). Individual values wiihin the normal range are notplotted.

Table 2. Maximal percent inhibition of IgA ANCAactivity in ELISA. obtained by co-incubaiion ofIgA ANCA-posilive sera with neutrophil extract

and with haemoglobin

HSPI23456789

1011

IgAN*SV*

Neutrophilextraet

8y52402430362661622133

4945

Haemoglobin

50020000203

80

* One serum oulor30 IgA nephropathy (IgAN)and one out of 40 systemic vasculitis (SV) sera wilhIgA ANCA positivity. Sec text. HSP. Henoch-Schonlein purpura.

Sera were diluted 1:40 and competitor proteinconcentration was 2 mg/ml. The inhibition wasdosc-depcndent in all cases.

52 N. Ronda et al.

66

51

56

Z9

20

Fig. 2. IgA ANCA Western btol obtained with Triton XlOO neutrophilextract after phorbol myrislate acetate (PMA) stimuialion. run in SDSPAGE under reducing conditions. Lanes I } were blotted with threeIgA ANCA Henoch-Schonlein purpura (HSP) sera, and show IgAreactivity wilh a 51-kD protein. Lanes 4 and 5 were blotted with normalsera. The other 12 normal and live IgA nephropathy control seraproduced no band (same appearance as in lane 5). The faini band ol'Ume4 is discussed in the te\t.

t(D

97 -a

6B

I 2 3Fig. 3. IgA ANCA Western biot obtained with fractions of theneulrophil extract. The neutrophil fraction containing solubilizcdmembranes and cytosolic material, obtained as supernatant aflcr acentrifugalion at 10()(H)A', was found lo contain ihe 51-kD protein. LaneI is ihe cooitiassie blue-stained nitrocellulose afler the protein transfer,Ljine 2 was blotted wilh a normal serum. Lane .^ was blotted wiili ;m IgAANCA-positive Henoch Scbonlein purpura (HSP) serum. IgA in thisserum reacted with the 51-kD protein and also with a 57-kD protein.The band al 68 kli in lanes 2 and 3 is non-spccilic and is due to direclbinding of the alkaline phosphatase-conjugated anti-hunian IgA.

showed significant IgA binding to the aniigcn-free wells. IgAANCA-positive HSP sera retained positivity with scrum dilu-tions ranging from 1:40 to 1:80. In all but one IgAN patientsIgA ANCA were not detectable, regardless of the disease phase.

All IgA ANCA-positive sera were also positive in the IgAlANCA SPIA; of these three HSP. one IgAN and one SV seraalso showed raised levels of IgA2 ANCA.

The inhibition of IgA ANCA activity obtained for eachpositive paiient by preinctibation of scrum with neutrophilextract and with haemoglobin is shown in Table 2.

Indirect immunqfluorescencePositive IgA IIF was obtained with all the positive seradescribed above, and produced a diffuse cytoplasmic staining,which did not have the granular appearance typically observedwith IgG ANCA IIF, Serum from one HSP patient producedboth cytoplasmic and perinuclear (PN) staining: this serum wasalso IgG ANCA-positive and produced a PN pattern in IgGIIK. Cytoplastnic staining was obtained by testing the two otherIgG ANCA-positive HSP sera in IgG-specific IIF.

IgA RF FUSANone of the HSP and IgAN sera showed IgA RF or IgG anti-IgA RF activity. IgA RF was found in one SV patient, positivefor both IgA and IgG ANCA: this patient was not considered asbeing positive for IgA ANCA.

To further exclude IgA RF interference in IgA ANCAmeasurements, sera from two IgA ANCA-positive HSP patientsand from the IgA RF-positive patient used as control werediluted l:i(). 1:20 and 1:40 and chromatographed on aSepharose column coupled to Protein A. which binds IgG, docsnot bind IgAl.and binds very weakly lgA2. IgA ANCA activityin the loaded and in the IgG-depletcd material was then assessedby ELISA, In the case of the positive IgA RF serum, a reductionin IgA ANCA activity of 29"-^ !, 55'V.i and 85"''.., compared withthe loaded material, was obtained by testing the eliluenlrespectively at 1:10. 1:20 and I :40. No difference was observedin IgA ANCA aetivity between the IgG-depleted and the loadedHSP sera at any of the dilutions.

Antigen-specific SPIATwo patients were found to have IgA and IgG anti-MPO

IgA ANCA in HSP 53

kD

68

29

('ig. 4. IgA ANCA Western blnl obiained wiili fractions of theiicuirophil extract. The ncutrDphil fraclion used in the cxpcrimetilsliown in Fig, } was further fraclionalcd by cenirifiigalion al 105000^and ihc same Henoch Schtinlcin purpura (HSP) and conirol sera as inlig, .1 were used for the immunoblot. Results obtained wilh ihesupernatanl. thai is soluble cytoplasmie material, are shown in lanes 1. 2and 3; results relative to the pellet. Ihat is the membrane-rich fratlion.are shown in lanes 4. 5 and 6. Lanes I and 4 show ihe coomassie blue-slained nitrocellulose after prolein transfer; the membrane-rich fraction(lane 4) is relatively depleted in prolein contenl. Lanes 2 and 5 wereiiicubilled wilh ihe healthy control serum and lanes 3 and 6 wilh the HSPpatient serum. The almost complete lack of reactivity of patient's IgAagainst ihe soluble cytoplasmie fraetion ofthe neutrophil extrnci and theconserved reactivity against the membrane-rich fraclion, relativelydepleleJ in prolein content, indicate ihat the followed procedureprovides a partially purified antigen preparation and that the 51-kDprotein recognized by ihe four strongest IgA ANCA-positive HSP serais probably membrane-associaled.

97

68

51

43

2 9 -

1 2 3 4 5Fig. 5. Western blot on total neiitrophil extract to test reaclivity of anti-fibroneclin antibodies. Lanes 1 and 2 were bloited with IgA ANCA-positive Henoch-Schonlein purpura (HSP) sera as positive controls.Lanes 3. 4 and 5 were bloited respectively with rabbit polyclonal anti-human fibroneclin, mouse monoclonal anti*human fibroncctm andmouse monoclonal anti-cellular fibroncclin. None ofthe anti-fibronec-lin antibodies reeognized the 51-kD prolein whieh is recognized by HSPIgA, The three antibodies reacted with a 25-kD protein in the neulrophilextract and two of them with a 4()-kD prolein, IgA from one of thepatients produced a faint band of reaclivily at 25 kD. Anii-tibronectinantibodies reacted with the same molecular species in experiments (notshown) performed using the membr:ine-rieh fraction of neulrophilextract, but the reactivity was much weaker. In this case HSP IgA whichhad weakly reacted with the 25-kD prolein did not produce anydetectable band al ihat molecular weight, whilst both HSP IgA stronglyreacted wilh the 51-kD prolein.

aniibodies (one HSP and one SV), NeJiher IgG nor IgA anii-PR3 aclivity was delected in the sera with IgA ANCA positivity.

Hestetn hint for IgA ANCAThe three strongest IgA ANCA-positive HSP sera reacted wilha 51-kD protein in the Triton XIOO neutrophil extract afterPMA stimulation, under reducing conditions (Fig. 2), The samereaciion was observed at a lesser degree of intensity by testingthe sera on the supernatant obtained after PMA slimulalion.Another IgA ANCA-posilive HSP serum reacted against the 51 -kD and against another protein of 57 kD (Fig. 3), A very faintband at 51 52 kD was produced by one normal serum. None ofthe other 13 normal and five IgAN conirol sera showed anyreactivity. Western blot performed wiih ihe various cellularfractions showed that the membrane-rich fraction, that is thepellet obtained afler centrifugation at 105000 g. is stronglyenriched in the 51-kD prolein recognized by IgA ANCA-positive HSP sera (Fig, 4), The three dJITerenl anti-libronectiniinlibodies tested by Western blot on a total neutrophil extractand on the membrane-rich fraction did not show any reactivityut 51 kD (Fig, 5), All three produced a band of reactivity at 25kDand twoof them a second band al 40 kD, IgA from one of theHSP sera tested produced a very faini band at 25 kD in Westernblot performed with total neutrophil extract (Fig, 5).

DISCUSSIONThe high prevalence of IgA ANCA in HSP (79%) but not inIgAN {yv

54 A'. Ronda et a].frequently recognized by IgG or IgM ANCA: only one HSPserum showed IgA and IgG reactivity against MPO; this serumalone produced both cytoplasmic and perinuclear staining inIgA-specific HF. None of the sera reacted against PR3- Thereactivity of the four strongest IgA ANCA-positive HSP seraagainst a 51-kD protein in the Western blot suggests that a newautoantigen might be the target of IgA ANCA in this disease.We have also included in Kig. 2 the Western blot obtained withthe only control serum, of 14 normal and live IgAN tested.which produced a faint band at 51-52 kD. If the specificity ofthis binding is ultimately shown to be the same as that obtainedwith HSP sera, the finding could be explained by the occurrenceof natural ANCA activity in the sera of some normal indi-viduals. Such activity has already been described [27] and is notsurprising, sinee autoantibodies of several specificities consti-tute a substantial part of normal circulating immunoglobulin.The Western blot linding that Ihe 51-kD protein reeognized inthe neutrophil extract by four HSP sera is present in themembrane-rich fraction, which is relatively depleted in proteincontent, and is almost completely absent from the cytoplasmicsoluble compartment, indicates that the IgA ANCA target isprobably a membrane-associated protein. Characterization ofthis antigen by amino acid sequence analysis is currently theobjective of a separate study.

One of the most controversial issues about IgA ANCA inIgA-related renal diseases is the possibility of obtaining falsepositive results in ELISA or immunoRuorescence tests, due tothe inereased levels of circulating IgA. or to physicochemicalchanges of circulating IgA in these patients, which couldincrease the ability of IgA to bind to several molecules through"non specific" interactions [28]. In particular it has been pointedout that, because fibroneetin is found in small amounts inneutrophil extracts [21,29] and because IgA-fibronectin com-plexes are increased in IgAN [30], IgA fibronectin complexescould account, at least partly, for the IgA ANCA reactivitydetected in IgAN and HSP [21]. We have performed Westernblot experiments to test whether IgA reactivity towards fibro-nectin could be responsible for the IgA ANCA activity that wedetected in HSP sera. Threedifferent anti-fibronectin antibodiesdid not react with the 51-kD protein in the neutrophil extractthat appears to be the major target for HSP IgA. All anti-fibronectin antibodies reacted with a 25-kD protein and two ofthem with a 40-kD protein, supporting previous studies indicat-ing that fibronectin fragments could be present in the neutrophilextract. We also found that one of the HSP sera tested produceda weak band of reactivity at 25 kD. Taken together, thesefindings support the view that some antibody speeies within theIgA circulating pool in HSP patients may be able to interactwith neutrophil-assoeiated fibronectin [21]. but also confirmthat within such pool other antibodies specifically associatedwith HSP are able to react with a neutrophil antigen, anddeserve to be further studied. A matter of debate eould now bewhether all these antibodies should be ealled IgA ANCA. Untilwe have more information about fibroneetin-reaetive anti-bodies, at present we call IgA ANCA the antibody activitywhich we detected specifically in HSP patients and whichappears to be directed against a 51-kD protein.

IgA ANCA are probably autoantibodies with a relativelylow affinity for the antigen or/and present in small amounts inihe circulation, as suggested by the fact that they can be detectedin ELISA and in immunofluorescence within a relativciv narrow

range of serum dilutions. Moreover, at least a subset of IgAANCA reaets with a neutrophil protein which is probably notabundant in the cells. For these reasons, and for ihc multiplecriteria that we set to define positivity in order to minimize therisk of false positive results, we believe that IgA ANCA are notyet useful for diagnosis of HSP. at least until the antigen isidentified. However, in the case of particularly aggressivedisease. IgA ANCA monitoring might soon be a useful tool forelinicians, as suggested by a case report recently published [31],in which IgA ANCA titres correlated strictly with diseaseactivity.

We have shown that IgA ANCA occur in a high percentageof adult patients with HSP, but not with tgAN or SV. IgAANCA specificity in most cases appeared to be different fromthe target reported to be most commonly recognized by IgGANCA, This information may contribute to an understandingof the pathogenesis of Henoch Schonlcni purpura and may beuseful for the classification of systemic vasculitides.

ACKNOWLEDGMENTSN,R. is a recipient of a gram from tlie Universily of Roma Tor VergaUi.Italy.

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