Clin Exp Immunol 1993; 94:440-446

Antibodies to proteinase 3 increase adhesion of neutrophilsto human endothelial cells

W.-J. MAYET & K.-H. MEYER ZUM BUSCHENFELDE L. Mecdicail Department, University of Main:,Mainz, Geranamy

(Acceptedt tbr publication 25 A ugust 1993)

SUMMARY

The detection of anti-neutrophil cytoplasmic antibodies (ANCA), especially those with specificity forproteinase 3, is important in the diagnosis and in monitoring disease activity of Wegener'sgranulomatosis and related vasculitides. An ubiquitous feature of all ANCA-associated acute

vascular injury is lytic necrosis. Adhesion of neutrophils to endothelium is a fundamental early step ofthe inflammatory response. Recently we were able to show that ANCA recognize their target antigen

(proteinase 3) translocated into the membrane of human endothelial cells. The aim of this study was

to investigate the effect of ANCA on the adhesion of neutrophils to human endothelial cells.Incubation of endothelial cells with affinity-purified antibodies to proteinase 3 (IgG- and F(ab')b-fractions) led to a marked increase of neutrophil adhesion, with a peak after 4 h and a rapid decreaseafter 8 h. This effect could be inhibited by preincubation of the endothelial cells with an antibody to

endothelial-leucocyte adhesion molecule-l (ELAM-l). Incubation with antibodies to proteinase 3also led to an increase of endothelial ELAM-I expression as measured in a cyto-ELISA and by flowcytometry. Our data demonstrate a direct effect of ANCA on neutrophil-endothelial interactions.The enhanced adhesion of neutrophils occurs time-dependently via induction of ELAM- I expression

on the surface of endothelial cells. Our data give a hint of an ANCA-mediated mechanism ofendothelial injury via induction of neutrophil adhesion to vascular endothelium in Wegener'sgranulomatosis and other ANCA-related vasculitides.

Keywords ANCA proteinase 3 adhesion endothelial cells vasculitis

INTRODUCTION

Adhesion of neutrophils to vascular endothelium represents anearly and essential event in acute inflammation. Neutrophil-mediated injury of human endothelial cells (HEC) is consideredto be an important mechanism in the pathogenesis of Wegener'sgranulomatosis (WG) and related vasculitides [1]. Antibodiesdirected against cytoplasmic antigens of neutrophils (ANCA),especially proteinase 3 (PR-3), are proven to be a useful clinicaltool in supporting the diagnosis or monitoring disease activity inWG. Several in vitro studies support the hypothesis that ANCAare more than an epiphenomenon and are intimately involved inthe pathogenesis of vasculitides. Recently it has been shown thatANCA can activate neutrophils in vitro and induce degranula-tion and production of oxygen radicals [2]. Furthermore, wehave been able to identify PR-3 expressed in the membrane ofHEC becoming accessible to anti-PR-3 antibodies [3]. The aimof the present study was to investigate the effect of affinity-

Correspondence: Professor Dr K.-H. Meyer zum Bilschenfelde,I. Med. Klinik und Polikinik der Johannes Gutenberg-UniversititMainz, Langenbeckstr 1, D-55101 Mainz, Germany.

purified anti-PR-3 antibodies on the adhesion of neutrophils inan in 1uitro system with cultured HEC.

PATIENTS AND METHODS

Serum s /mplesSerum samples were obtained from 1 85 donors. Fifty sufferedfrom clinically active WG. The diagnosis was established on thebasis of classic symptoms and the typical histological findings inbiopsy specimens as described earlier [4]. Sera of 100 healthyblood donors (H BD), 25 patients with systemic lupus erythema-tosus (SLE), five patients with Sharp syndrome and five patientswith Sj6gren's syndrome (SS) served as controls.

Antibody testingAll WG sera were tested for anti-PR-3 antibodies by immuno-fluorescence technique (IFT) on fixed neutrophils [5], ELISAand Western blot. Several antigen preparations served asantigens: alpha-extract of human neutrophils [6], purified PR-3[7], myeloperoxidase, cathepsin G and elastase [8]. ELISAs wereperformed as described earlier [9]. PAGE and Western blottingwere performed as described previously [10,11]. In addition, allsera were screened with routine methods for other antibodyspecificities [10].

440

ANCA increase neutrophil adhesion to endotlhelial cel 441

Purification of anti-PR-3 antibodiesIgG was prepared from 10 monospecific anti-PR-3 antibody-positive WG sera by ammonium sulphate precipitation and ionexchange chromatography on DEAE-Sephadex (Pharmacia,Uppsala, Sweden). Anti-PR-3 antibodies were affinity-purifiedas described earlier [12] using purified PR-3. F (ab')2 fragmentswere prepared as previously described [4]. For inhibitionexperiments affinity-purified anti-PR-3 antibodies were mixedwith different extracts of human neutrophils. an extract ofHEp2-cells (control), affinity-purified PR-3 antigen and PR-3purified as described by Kao et al. [7] v/v diluted to 0-1 mg/mlprotein concentration in PBS and incubated on a rotator for I hat 37 C and 12 h at 4 C. The mixture was centrifuged at 30 000gfor 15 min at 4 C and the supernatants kept as absorbedmaterial. An anti-PR-3 specific B cell clone has been establishedand characterized earlier [13]. Antibody preparations weretested for endotoxin using a commercially available E-toxateassay (Sigma, Deisenhofen, Germany).

Purification of anti-Ro (SS-A), anti-La (SS-B) and anti-RNPantibodiesAntibodies to Ro (SS-A). La (SS-B) and RNP were purified andcharacterized as described previously [12].

Preparation of cell extractsAn extract (alpha-fraction) of human neutrophils was preparedas described by Rasmussen et al. [6] and Savage et al. ('acid'extract) [14]. An extract of HEp2-cells was prepared as de-scribed earlier [4]. Total extracts of cytokine-treated HEC wereprepared according to Albeda et al. [15]. Only cell cultures freeof fibroblasts or monocytes obtained after several passages wereused for these experiments.

Purification of PR-3PR-3 was purified as described by Kao et at. [7] and affinitypurified as described by Liudemann et al. [11 ] using an extract ofgranulocytes.

Isolation and culture of human endothelium (ellsHEC were isolated according to the method of Jaffe et al. [16]and cultured under standard conditions [17]. These cells wereused for further experiments between passages 4 and 6. Cells of10 donors were pooled to exclude the influence of blood groupantigens, and morphology was confirmed by phase contrastlight microscopy showing the typical cobblestone monolayerappearance of cells. Purity of culture was tested with antibodiesto factor VIII-antigen and Ulex lectine [18].

HEC were passaged on gelatine-coated culture slides (Lab-Tek; Miles Scientific, Naperville, IL) or Primaria culture dishes(Falcon, NJ) and fixed with ethanol 96',, methanol (abs) orparaformaldehyde 3 71% (-20 C for 10 min) [12]. Tumournecrosis factor-alpha (TNF-7) (3 ng/ml; Boehringer, Mann-heim, Germany) was added to the medium before fixation to testthe influence of this cytokine on antigen expression. Appro-priate cytokine concentrations were determined by testingdilutions from 0-001 to 100 ng/ml, and a polyclonal anti-TNFantibody (Camon, Wiesbaden, Germany) was used to blockTNF-specific effects in some tests. Cells on Primaria culturedishes were left unfixed. Cells were incubated with purified IgG,F(ab')2 fragments, and the supernatant of the PR-3-specitic Bcell clone Ho3 diluted 1:40 in PBS or an anti-ELAM-1 antibody

(No. 0805; Dianova, Hamburg, Germany) for 1-120 min in ahumid chamber at room temperature or 4 C. After extensivewashing with PBS the cells were incubated with the secondantibody, FITC-conjugated anti-human IgG (F-5512; Sigma,Deisenhofen, Germany). For detecting surface expression ofantigens, all experiments were done with unfixed live cells asdescribed earlier [10]. Flow cytometric analysis of HEC wasperformed as described by Furukawa et al. [19].

Cyto-ELISAs with unfixed cytokine-treated HEC wereperformed as described by Frampton et al. [20]. with minormodifications. To determine specific antibody binding, experi-ments were performed with HEC preincubated with heat-aggregated human IgG to block Fc receptors [21].

Isolation of neutrophilsNeutrophils were isolated as recently described by Toothill et al.[22].

Adhesion assa,55HEC ( 104 well) were seeded in wells of 6 41 mm diameter andleft for 24 h in a CO, atmosphere. The confluent HEC wereincubated with sera of WG patients (1:80 diluted in medium199), affinity-purified anti-PR-3 antibodies (5 pgrml). the super-natant of the human anti-PR-3-specific B cell clone Ho3 andrecombinant TNF-t (1 ng, ml; Boehringer) for 4 h. Medium 199,purified anti-Ro (SS-A). anti-La (SS-B) and anti-RNP anti-bodies (5 jig ml). a serum pool of 100 HBD and PBS served ascontrols. In addition. HEC preincubated with an anti-endothe-

*2 -

*.I_

O'8 t-

i'iNd. 0-6>_

a

C

00

0

0 -4

01Fig. 1. Optical densities (OD) of F(ab')2 fragments of affinity-purifiedanti-proteinase 3 (PR-3) antibodies. ELISA with purified PR-3 asantigen. Data including mean + s.d. are given in Table 1. 0. Ten patientswith Wegener's granulomatosis (WGI 10): 0. anti-PR-3 antibodyclone Ho3 (PR-3-EBV); *, mean of 100 healthy blood donors +s.d.

441

W.-J. Mayet & K.-H. Meyer zum Biischenfelde

A a c. _

29 _

Fig. 2. Western blot with alpha-fraction of human neutrophil granulocytes. (A) Ten F(ab')2 fragments of affinity-purified anti-proteinase 3 (PR-3) antibodies ofpatients with Wegener's granulomatosis. Reaction at 29 kD. (B) Reaction ofanti-PR-3 antibody Ho3.(C) Reaction of four sera of healthy blood donors (controls).

0 4

Time (hWFig. 3. Kinetics of adhesion of neutrophils to human endothelial cells.Cyto-ELISA with unfixed cells. Measurements at 0, 1, 2, 24 and 48 h ofincubation with different purified antibodies. One mark represents themean of three measurements (data including mean + s.d. are given inTable 2).WG 1 -5, F(ab')2 fragments of purified anti-proteinase 3 (PR-3)antibodies; PR-3-EBV, anti-PR-3 antibody Ho3; TNF-a, effect ofTNF-a; WG I +a-ELAM, affinity-purified anti-PR-3 antibody+anti-ELAM-l antibody. 0, WGI; A, WG2; 0, WG3; v, WG4; *, WG5;A, PR-3-EBV; *, TNF-a; - - , WGI +a-ELAM.

lial-leucocyte adhesion molecule- I (ELAM- I) antibody (1: 500,30 min; Dianova) were also investigated. After a washing step,HEC were incubated with freshly prepared human neutrophils200 ,l (2 x 106/ml) for 15-30 min at 37°C. Non-adherent cellswere soaked off and every well was washed again. A 0-250/0Bengal red solution was added (5 min at 24"C), and after twoadditional washing steps adherent neutrophils were lysed withethanol/PBS (v,v). After 30 min the optical density (OD) was

measured in a photometer (570 nm). OD ofHEC alone served as

control [23].

0.8

0 6

0

o 0*4

0-2

0 4 8 12 16 20 24 28 32 36 40 44 48Time (h)

Fig. 4. Kinetics of adhesion of neutrophils to human endothelial cells.Cyto-ELISA with unfixed cells. Measurements at 0, 1, 2, 24 and 48 h ofincubation with different purified antibodies. One mark represents themean of three measurements (data including mean + s.d. are given inTable 1). Ro 1-3, La, RNP, WG 1: F(ab')2 fragments of purifiedantibodies; TNF-a, effect of TNF-a. 0, Rol; A, Ro2; 0, Ro3; v, La;*, RNP; A, WGI; *, TNF-a.

RESULTS

Ten WG sera were monospecifically positive for anti-PR-3antibodies as determined by IFT on human neutrophils (C-ANCA+; titres 1:320-1:640), by ELISAs with alpha-fractionand 'acid' extract of human neutrophils and total extract ofcytokine-treated HEC, by Western blot (reaction at 29 kD) andother routine methods [10] (data not shown). F(ab')2 fragmentsof the affinity-purified antibodies reacted positively in an ELISA(Fig. 1) and in Western blot (Fig. 2). Antibody reactivity couldbe blocked by incubation with purified PR-3 antigen (affinitypurified as well as KAO-preparation) as measured by ELISAand determined by Western blot (data not shown). Antibodyreactivity could not be inhibited by preincubation with extractsof HEp2 cells. In addition, four sera of patients with SS and one

442

(

ANCA increase neutrophil adhesion to endothelial cells

Table 1. Data of Fig. 4

WGI WG2 WG3 WG4 WG5 WG6 WG7 WG8 WG9 WGIO PR-3-EBV

1-13 092 098 086 1-03 087 073 097 1 10 089 090 OD1 03 0 95 0 87 0-89 1-22 0-73 0 71 1-12 1 31 0-92 0-87 OD1.09 0-87 096 0-78 1 19 0 80 0-68 1 10 1-02 098 0 83 OD1-08 0-92 094 084 1-15 080 071 106 1-14 093 087 M004 004 005 0 05 0-08 0-06 002 0 07 0 12 0-04 003 SD

Table 2. Data of Fig. 3

WGI WG2 WG3 WG4 WG5 Rol Ro2 Ro3WGI +

La RNP TNF-a PR3-EBV a-ELAM

0-184 0093 0-1160 200 0-165 0-1790 218 0-199 0 2130 201 0 152 0 1690-017 0-054 0 049

0417 0-311 0-4690 394 0-396 0-3060 423 0-334 0-2980411 0347 03580015 0044 0096

0-621 0 498 0-3870633 0564 04110 601 0 503 0 4050-618 0 522 0 4010-016 0 037 0-012

0516 0502 0-3870 587 0 477 0-3940605 0516 03650-573 0 498 0-3820 050 0-020 0 015

0-329 0 477 0-2880366 0326 03170-398 0-365 0 2590 364 0-389 0 2880 034 0-078 0-029

0-156 0273 02110-294 0-200 0 2140243 0-193 0 1960 231 0-222 0-2070070 0044 009

serum of a patient with Sharp syndrome were found to bepositive for anti-Ro (SS-A), anti-La (SS-B) and anti-RNPantibodies. Antibodies of these sera were purified and served as

controls. Antibody preparations diluted to the highest concen-

trations used in the experiments were free of endotoxin as

determined by a limulus amoebocyte lysate assay.

Adhesion of neutrophilsIncubation of HEC with purified anti-PR-3 antibodies incontrast to anti-Ro (SS-A), anti-La (SS-B) and anti-RNPantibodies (controls) led to a marked increase of neutrophiladhesion starting after 30 min antibody incubation, and a peak

after 4 h followed by a gradual decrease within the next 44 h(Figs 3 and 4; Table 1). This effect could be partially inhibited bypreincubation of HEC with an antibody to ELAM-1 (Fig. 3).Treatment of HEC with TNF-a caused similar kinetics, with a

transient increase of neutrophil adhesion peaking after 4 h.TNF-ac (control) had no visible effect on the integrity of the HECmonolayer and had no influence on the capacity ofHEC to takeup rose Bengal stain.

Induction of adhesion moleculesTreatment of HEC with TNF-a led to a maximal membraneexpression of ELAM-I after 4 h (Fig. Sa). Incubation of HEC

443

Time(h)

0-1430-186

0 02000-1760 030

04860 434

2 04010 4400043

06430-639

4 0-6420-6410-002

0 5490-626

8 057805840 040

04650422

24 0 3860-4240-040

0-1580-165

48 0 1380 1540014

0-109 0 0670-123 0 0630-296 0-0560-176 0 0620-104 0005

0 266 0 0740 290 0-0680 309 0-0890-288 0-0770021 0009

0 504 0 0920-478 0-0870-525 0-0970 502 0-0920023 0004

0 506 0-0900 479 0-0860-483 0-0910-489 0 0890014 0003

0311 00740-283 0-0770 259 0 0830 284 0-0780036 0004

0-243 0 0610-216 0 0480-249 0-0790 236 0-0630 017 0-010

0 13400980 1060-11300 15

0-1260-1110 1230 1200006

0 1380-1420-1310-1370-005

0-0260-1820 1400-1940 050

0-1380-1880-2300-1850 090

02450-2830-2090-2460 030

0-0980091008700920-004

0-0880-089009700910 004

00980 09900910 0960 004

0-1880-1920-1830-1880 004

0-2240-2830-10402040070

0 1630 1280-1130-1350021

0-190 02000-187 0-1980-182 0-1750-186 0-1910003 0011

0 165 00990 153 0 0870-186 00800-168 0-0890014 0008

0 265 0-0750-233 0-1030-234 0-0980 244 0-0920.015 0-012

0230 02310 201 0 1990-198 02040201 02110-014 0-014

0 187 0-1850 226 0 1260-195 0 1330-203 0 1480017 0026

0-270 0-0860-179 0-1080-186 0-1010 212 0-0980-041 0 009

0-3660-2730 20302810-082

0 3870-32203510 3530 032

08770-8460 8020-8420038

0 4660 456038404360-044

0-38803240 3170 3930039

0-1940-2360 2490 2260 029

0 0940-0860-1880-1230056

0 1860-1140-1960-1650045

0-3220-2940 3490-3550 036

0 1650 1470-1240 1450-020

0*1000 0980 0900 0960005

0-0360-1630 0840 1140042

0 1280-1530 1860-1550-029

0-2860-2530 2960 2780021

0 38903940 3830-3880005

0 3290-3160 3060 3170012

0254024402100-2360-023

0 1360 1260-1450-1360 009

ODODODMSD

ODODODMSD

ODODODMSD

ODODODMSD

ODODODMSD

ODODODMSD

W.-J. Maqyet & K.-H. MeYer zumn Biischenfelde

0*6

O0

0-4

0 *2

0

0 4 8 12 16 2C 24 28 32 36 40 44 48

Time ( h)

Fig. 5. Kinetics of endothelial ELAM-I expression. Cyto-ELISA withunfixed human endothelial cells. Measurements at 0. 2. 4, 24. 48 h ofincubation with different purified antibodies. One mark represents themean of three measurements. WGl 10, F(ab')2 fragments of purifiedanti-proteinase 3 (PR-3) antibodies; TNF-c, effect of TNF-o; NS-pool,serum pool of healthy blood donors. (a) E. WGI; A, WG2; 0, WG3;v, WG4; *, WG5; 0* TNF-t. (b) 0, WG6; A, WG7; 0. WG8; v. WG9;*, WGIO; 0, NS-pool.

Fig. 6. Indirect immunofluorescence of unfixed human endothelial cellsalfter incubation with purified anti-proteinase 3 (PR-3) antibody WGI(original magnification x450). Visualization of ELAM-I expressionwith an FITC-conjugated anti-ELAM-I antibody.

0 200 400 600 8001000 0 200 400 600 800 1000128 64

-(a) i(b)

'0 10410 110 0

o 102 103 4 0 10 102 103 104Fluorescence I Fluorescence

Fig. 7. Flow cytometric analysis ofhuman endothelial cells. (a) Reactionof an anti-ELAM-l antibody with untreated cells. No ELAM-lexpression. (b) Reaction of an anti-ELAM-l antibody after preincuba-tion of endothelial cells with affinity-purified anti-proteinase 3 (PR-3)antibodies. Induction of ELAM-1 expression.

with affinity-purified anti-PR-3 antibodies resulted in anincreased membrane expression of ELAM-1, with a maximum

after 4 h as determined by cyto-ELISA (Fig. 5ab), IFT (Fig. 6)and flow cytometric analysis (Fig. 7). Kinetics of ELAM-1expression after incubation with anti-PR-3 antibodies was

similar to that obtained after incubation of HEC with TNF-a(control). Incubation of HEC with a serum pool of HBD(control) had no significant effect.

DISCUSSION

The adherence of neutrophils to vascular endothelium and theirsubsequent emigration is a hallmark of acute inflammation.Leucocytes are capable of producing a number of products thatcan potentially cause cell injury or modify cell function.Consequently, many investigators have proposed a causalrelationship between leucocyte accumulation at sites of inflam-mation or immune reaction and associated vascular and tissueinjury. A major factor involved in leucocyte recruitment intoinflammatory tissues is thought to be the expression ofcytokinc-inducible adhesion molecules on vascular endothelial cells.There is increasing evidence that a number of endothelialadhesion molecules are involved in preferentially bindingparticular leucocyte types [24,25]. Immunohistological studiescan demonstrate these molecules in tissue sections [26]. Closeapproximation of the leucocyte to the endothelium allows short-lived mediators such as oxidants to act.

The two adhesion molecules that have been characterizedmost fully are intercellular adhesion molecule-I (ICAM-I) [24]and ELAM-I [25]. The surface expression of ICAM-I is up-regulated on HEC by IL-I, TNF, lipopolysaccharide (LPS) orinterferon-ganmma (IFN-;) [27]. ICAM-I is a ligand for theleucocyte adhesion molecule LFA- (CDI Ia/CDl8) [28] and islikely to be involved in the adhesion of both neutrophils andlymphocytes [22] to cytokinc-activated endothelium. In contrastto ICAM-l. ELAM-I is found only on activated HEC and itsexpression is induced by IL-I, TNF or LPS but not by IFN-;,[25,28]. Experiments with MoAbs to ELAM-I cDNA-trans-fected cells indicate that ELAM-1 is a selective adhesionmolecule with the ability to bind neutrophils rather thanlymphocytes [24,29].

ELAM-I is not basally expressed on unstimulated endothe-lial cell surface, but is rapidly induced by a large number of

444

(1)n

ANCA increase neutrophil adhesion to endothelial cells 445

cytokines [30]. ELAM-1 expression is initiated 30 min afterexposure to these cytokines, its adhesive action peaking 4 h afterstimulation and declining to background levels by 24 h despitecytokine presence [30].

An ubiquitous feature of all ANCA-associated acute vascu-lar injury is lytic necrosis, and the adhesion of neutrophils toendothelium is thought to be a fundamental early component ofthe inflammatory response [31]. Evidence exists that both theautoantigen and ANCA participate in the pathogenesis of atleast the group of ANCA-associated vasculitides [32]. Recentlyit was shown in vitro that F(ab)2 fragments from ANCAincrease neutrophil adhesion to HEC augmented by prestimula-tion with TNF-z [33]. Savage et al. [34] reported binding of PR-3and myeloperoxidase added to HEC, turning these cells intotarget structures for ANCA.

In this study we have investigated the effects of purified anti-PR-3 antibodies on HEC. Anti-PR-3 antibodies, as well asTNF-o induced ELAM-l expression on HEC, with a peak after4 h and a rapid decrease during the following hours. Adhesion ofneutrophils was observed with similar kinetics.

Our data demonstrate a direct effect of anti-PR-3 antibodieson neutrophil-endothelial interactions, i.e. anti-PR-3 antibodiescan act on vascular endothelium to alter its fundamental surfaceproperties related to neutrophil adhesion. The enhanced neutro-phil adhesion occurs time-dependently via induction of ELAM-I expression on the surface of HEC. The exact mechanism ofanti-PR-3 antibody-triggered induction of ELAM-I expressionis not known. Recently. we were able to demonstrate PR-3previously considered myeloid-specific in a human renal cancerline [35] and in HEC. Cytokines like TNF-x, IL-I and IFN-;induce an increased PR-3 expression in the cytoplasm of HEC.PR-3 is also expressed on the surface ofH EC, thereby becomingaccessible to the respective antibody [3]. However, we consider itlikely that anti-PR-3 antibody binding to PR-3 on endothelialcell surface induces an activation change within the endothelialcells. Apparently, some form of signal transduction ensues afteranti-PR-3 antibodies bind to their antigens on the HEC plasmamembrane. Regarding these data, we conclude that vascularinjury in ANCA-associated vasculitis disorders may be trig-gered by anti-PR-3 antibodies, which in addition may directlyactivate neutrophils, as suggested by Falk et al. [2]. Closeadhesion between neutrophils and endothelial cells may allowthe formation of a sequestered microenvironment which is lessaccessible to the protective effects of proteinase inhibitors (i.e.71-antitrypsin for PR-3) [36]. It is evident that the inhibition ofneutrophil adherence to microvascular endothelium wouldprevent influx of neutrophils into tissue. This, in turn, leads to areduction in tissue damage mediated by polymorphonuclearleucocytes. Our data suggest an ANCA-mediated mechanism ofendothelial injury via induction of neutrophil adhesion tovascular endothelium in WG and other ANCA-related vasculi-tides. Cytokine antagonists may block endothelial reorganiza-tion and perhaps injury, as well as inhibit endothelial contribu-tions to adhesion and leucocyte activation. Perhaps the mostpromising avenue is local blockage of endothelial cell receptors(ICAM-l and ELAM-l), since localized treatment may causeless adverse effects than would systemic inhibition of neutrophilfunction [30]. However, many of these steps may prove to haveunintended consequences. Such approaches will need to betested both in animal models and in careful clinical investiga-tions [37].

ACKNOWLEDGMENTS

We are grateful for the expert technical assistance of Ms Britta Kuhn.We thank Professor Dr F. Peters and the staff of the St. HildegardisHospital Mainz for collecting umbilical cords, and we thank Dr IlkaHelmreich-Becker for reviewing the manuscript.

REFERENCES

1 Churg J. Churg A. Idiopathic and secondary vasculitis: a review.Mod Pathol 1989; 2:144-60.

2 Falk RJ. Terrel RS. Charles LA. Jennette JC. Anti-neutrophilcytoplasmic autoantibodies induce neutrophils to degranulate andproduce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990;87:4115 -9.

3 Mayet WJ, Csernok E, Szymkowiak, Gross WL, Meyer zumBuschenfelde KH. Human endothelial cells express proteinase 3, thetarget antigen of anticytoplasmic antibodies in Wegener's granulo-matosis. Blood 1993; 82:1221-9.

4 Nolle B. Specks U. Ludemann L. Rohrbach MS. DeRemee RA,Gross L. Anticytoplasmic autobodies: their immuno-diagnosticvalue in Wegener's granulomatosis. Ann Intern Med 1990: 111:28.

5 van der Woude FJ. Lobatto S. Permin H et al. Autoantibodiesagainst neutrophils and monocytes: tool for diagnosis and marker ofdisease activity in Wegener's granulomatosis. Lancet 1985; ii:425 -9.

6 Rasmussen N. Sjolin C, Isaksson B, Bygren P. Wieslanider J. AnELISA for the detection of anti-neutrophil cytoplasm antibodies(ANCA). J Immunol Meth 1990; 127:139-45.

7 Kao RC, Wehner NG, Skubitz KM, Gray H. Hoidal J. Proteinase 3.A distinct human polymorphonuclear leukocyte proteinase thatproduces emphysema in hamsters. J Clin Invest 1988; 82:1963-73.

8 Mayet WJ. Hermann E. Finsterwalder J. Rieder H. Poralla T. Meyerzum Bu3schenfelde KH. Antibodies to cathepsin G in Crohn'sdisease. Eur J Clin Invest 1992; 22:427 33.

9 Mayet WJ. Bachmann M, Pfeifer K, Schroder HC, Muller WEG,Meyer zum Buischenfelde KH. A monoclonal Ro-antibody and theserum of a Ro-positive patient with subacute cutaneous lupuserythematosus (SCLE) react with basal layers of human epidermis.Eur J Clin Invest 1988: 18:465-71.

10 Mayet WJ. Hess G. Gerken G. Rossol S. Voth R. Manns M, Meyerzum Buischenfelde KH. Treatment of chronic type B hepatitis withrecombinant alpha-interferon induces autoantibodies not specificfor autoimmune chronic hepatitis. Hepatology 1989; 10:24-28.

11 Lidemann J. Utecht B. Gross WL. Detection and quantitation ofantineutrophil cytoplasm antibodies in Wegener's granulomatosisby ELISA using affinity-purified antigen. J Immunol Meth 1988:114:167 70.

12 Bachmann M, Mayct WJ, Schroder HC, Pfeifer K, Meyer zumBuischenfelde KH, Muller WEG. Association of La and Ro antigenswith intracellular structures in HEp-2 carcinoma cells. Proc NatlAcad Sci USA 1986: 83:7770- 4.

13 Mayet WJ, Hermann E. Kiefer B. Lehmann H. Manns M. Meyerzum Bischenfelde KH. In ritro production of anti-neutrophilocytecytoplasm-antibodies (ANCA) by Epstein Barr virus-transformedB-cell lines in Wegener's granulomatosis. Autoimmunity 1991;11:13 19.

14 Savage CO. Winearls CG. Jones S. Marshall PD. Lockwood CM.Prospective study of radioimmunoassay for antibodies againstneutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet1987; i:1389 92.

15 Albeda SM, Oliver PD. Romer LH, Buck CA. EndoCAM: a novelendothelial cell cell adhesion molecule. J Cell Biol 1990; 110:1227-37.

16 Jaffe EA, Nachmann RL, Becker CG. Culture of human endothelialcells derived from umbilical veins: identification by morphologiccriteria. J Clin Invest 1973: 52:2745-56.

446 W.-J. Mayet & K.-H. Mever zun Biischenf/lde

17 Thornton SC, Mueller SN, Levine EM. Human endothelial cells: useof heparin in cloning and long-term serial cultivation. Science 1983;222:623-5.

18 Bevilaqua MP, Pober JS, Majeau GR, Fiers W, Cotran RS,Gimbrone MA. Recombinant tumor necrosis factor induces procoa-gulant activity in cultured human vascular endothelium. Proc NatlAcad Sci USA 1986; 83:4533-6.

19 Furukawa F, Kashihara-Sawami M, Lyons MB. Norris DA.Binding of antibodies to the extractable nuclear antigens SS-A/Roand SS-B/La is induced on the surface of human keratinocytes byultraviolet light (UVL): implications for the pathogenesis of photo-sensitive cutaneous lupus. J Invest Dermatol 1990; 94:77 85.

20 Frampton G. Jayne DRW, Perry GJ, Lockwood CM, Cameron JS.Autoantibodies to endothelial cells and neutrophil cytoplasmicantigens in systemic vasculitis. Clin Exp Immunol 1990; 82:227- 32.

21 Rekvig OP, Hannestad K. Certain polyclonal antinuclear antibodiescrossreact with the surface membrane of human lymphocytes andgranulocytes. Scand J Immunol 1977; 6:1041-7.

22 Toothill VJ, Van-Mourik JA, Niewenhuis HK, Metzelaar MJ,Pearson JD. Characterization of the enhanced adhesion of neutro-phil leukocytes to thrombin-stimulated endothelial cells. J Immunol1990; 145:283 91.

23 Gamble JR, Vadas M. A new assay for the measurement of theattachment of neutrophils and other cell types to endothelial cells.J Immunol Meth 1988; 109:175-84.

24 Dustin ML, Springer TA. Lymphocyte function-associated antigen-I (LFA-I) interaction with intercellular adhesion molecule-l(ICAM-1) is one of at least three mechanisms for lymphocyteadhesion to cultured endothelial cells. J Cell Biol 1988; 107:321-31.

25 Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, GimbroneMA. Identification of an inducible endothelial-leukocyte adhesionmolecule. Proc Natl Acad Sci USA 1987; 84:9238-42.

26 Muller GA, Marrovic-Lipowski J, Muller CA. Intercellular ad-hesion molecule-l expression in human kidneys with glomerulo-nephritis. Clin Nephrol 1991; 36:203 -8.

27 Pober JS, Bevilaqua MP, Mendrick DL, Lapierre LA, Fiers W.Gimbrone MA. Two distinct monokines, interleukin I and tumornecrosis factor each independently induce biosynthesis and transientexpression of the same antigen on the surface of cultured humanvascular endothelial cells. J Immunol 1986: 136:1680--4.

28 Kyan-Aung U, Haskard DO, Poston RN, Thornhill MH, Lee TH.Endothelial leukocyte adhesion molecule-I and intercellular ad-hesion molecule- I mediate the adhesion of eosinophils to endothelialcells in itr(o and are expressed by endothelium in allergic cutaneousinflammation in vitro. J Immunol 1991; 146:521-8.

29 Ikuta S, Kirby A, Shenton BK, Givan AL, Lennard WJ. Humanendothelial cells: effect of TNF-alpha on peripheral blood mono-nuclear cell adhesion. Immunology 1991; 73:71-6.

30 De la Ossa JC, Malago M, Gewertz BL. Neutrophil-endothelial cellbinding in neutrophil-mediated tissue injury. J Surg Res 1992;53:103 7.

31 Gamble RJ, Harlan JM, Klebanoff SJ. Lopez AF, Vadas MA.Stimulation of the adherence of neutrophils to umbilical veinendothelium by human recombinant tumor necrosis factor. ProcNatl Acad Sci USA 1985; 82:8667 71.

32 Hagen EC, Ballieux BE, van Es LA, Daha MR, van der Woude FJ.Antineutrophil cytoplasmic autoantibodies: a review of the antigensinvolved, the assays and the clinical and possible pathogeneticconsequences. Blood 1993: 81:1996 2002.

33 Keogan MT, Rifkin 1, Ronda N, Lockwood CM, Brown DL.Antineutrophil cytoplasm antibodies (ANCA) increase neutrophiladhesion to altered human endothelium. In: Gross WL, ed. ANCAassociated vasculitides: immunodiagnostic and pathogenetic valueof antineutrophil cytoplasmic antibodies. London: Plenum Press, inpress.

34 Savage COS, Pottinger BE, Gaskin G, Pusey CD, Pearson JD.Autoantobodies developing to myeloperoxidase and proteinase 3 insystemic vasculitis stimulate neutrophil cytotoxicity toward culturedendothelial cells. Am J Pathol 1992: 141:335 43.

35 Mayet WJ. Hermann E, Csernok E. Knuth E, Poralla T, Gross WL,Meyer zum B13schenfelde KH. A human cancer line as a new antigensource for the detection of antibodies to cytoplasmic and nuclearantigens in sera of patients with Wegener's granulomatosis. JImmunol Meth 1991; 141:57-68.

36 Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-76

37 Pober JS. Cotran RS. The role of endothelial cells in inflammation.Transplantation 1990: 50:537 44.