American Journal of Pathologv, Vol. 149, No. 1, July 1996Copynrght © American Society for Investigative Pathologv

Role of Neutrophils in the Pathogenesis ofExperimental Vasculitis

Faieza J. Qasim,* Peter W. Mathieson,*Fujiro Sendo,t Sathia Thiru,* andDavid B. G. Oliveira*From the Departments ofMedicine * and Pathology,tUniversity of Cambidge, Cambnidge, United Kingdom andIshikawa University,t Ishikawa, Japan

In the Brown-Norway rat, mercuric chloride(HgCI2) induces an autoimmune syndrome char-acterized by high IgE levels. There is widespreadnecrotizing leukocytoclastic vasculitis involvinglung, skin, mucous membranes, pancreas, liver,and gut, with tissue injury being most marked inthe cecum. As in systemic vasculitis in man, thereare neutrophils at the site of tissue injury andthe animals develop anti-neutrophil cytoplasmicantibodies, which in the Brown-Norway rat aredirected against myeloperoxidase. To determinewhether neutrophils are involved in the patho-genesis of the vasculitis, we have used a mono-clonal antibody that was reported to deplete neu-trophils in other rat strains. Rats treated withHgCI2 received antibody by intravenous injectionat various timepoints. Serial blood samples weretaken for neutrophil counts and to assay foranti-myeloperoxidase and IgE antibodies. Theguts of animals kiled after antibody therapywere scoredfor vasculitic changes and neutro-phil infiltrate. RP3 (but not the control antibodyMAC6) was shown to bind to Brown-Norway ratneutrophils and to block glycogen-induced influxof neutrophils into the peritoneum. When givenat peak disease, RP3 caused a dose-dependentreduction in tissue injury with a marked reduc-tion in circulating blood neutrophil numbers andin tissue neutrophil infiltrate. RP3 treatment didnot affect the rise in titer of IgE and anti-my-eloperoxidase antibodies. The data presenteddemonstrate that in this model neutrophils arenecessaryfor the induction ofvasculitis and thatthe degree of vasculitis correlates with neutro-phil number. To our knowledge, this study is thefirst to provide direct evidence for a role for

neutrophils in vasculitis. We suggest that anti-bodies directed against neutrophils, especialy ifthey deplete neutrophils, may be useful in thetherapy ofvasculitis in man. (AmjPathol 1996,149:81-89)

In man, the systemic vasculitides are a group ofautoimmune diseases characterized by necrotizingvasculitis, which may be associated with circulatingautoantibodies. The diseases are classified accord-ing to the size of vessel, pattern of organ involve-ment, and presence of granulomata.' Small vesselvasculitides are associated with antibodies againstneutrophil cytoplasmic antigens (ANCAs),2 andthese are predominantly anti-proteinase-3 (anti-RP3)or anti-myeloperoxidase (anti-MPO).3'4 The patho-genesis of these diseases is unclear, although indi-rect evidence exists for a role for ANCA antibodies(titer correlates with disease activity5 and these an-tibodies may activate neutrophils in vitro6'7). Infectionmay also play a role in the etiology of systemicvasculitis; relapse may be associated with intercur-rent infection,8 and data from uncontrolled studiessuggest that antibiotics may help to induce andmaintain remission.9 Effective treatment of vasculitisinvolves the use of broad-spectrum immunosuppres-sive agents and may be associated with side effectssuch as bone marrow toxicity and opportunistic in-fection.10

Neutrophils are implicated in the pathogenesisof systemic vasculitis by their presence at the siteof tissue injury, but there is little direct evidencethat they play a primary role. Neutrophils, attractedto an involved site by chemotactic cytokines suchas interleukin (IL)-8 that have been induced by

Faieza Qasim is a Medical Research Council Training Fellow. PeterMathieson is a Medical Research Council Clinician Scientist. Thiswork was supported in part by the Addenbrooke's Kidney PatientsAssociation.

Accepted for publication March 11, 1996.

Address reprint requests to Dr. Faieza J. Qasim, Department ofMedicine, Level 5, Addenbrooke's Hospital, Hills Road, CB2 20Q,Cambridge, UK.

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infection or injury, can be activated by inflamma-tory mediators (for example, complement compo-nents, cytokines, and antibodies) and cause dam-age by release of proteolytic enzymes andreactive oxygen species.11-14 The precise mech-anism involved in tissue injury could be studiedmore easily in an animal model than in man as amodel system allows controlled manipulation ofindividual putative pathogenic factors. In theBrown-Norway (BN) rat, mercuric chloride (HgCI2)induces an autoimmune syndrome15 with wide-spread tissue injury and, in particular, necrotizingleukocytoclastic vasculitis in the gut.16 There arevasculitic lesions in many organs including pan-creas, skin, and liver, and granulomata are seen inthe lung.17 Lesions are seen within 24 hours of thefirst injection of HgCI2 and maximal tissue injuryoccurs 2 weeks later.17 There are analogies tosystemic vasculitis in man; tissue injury can beameliorated by antimicrobial treatment18 and cir-culating ANCAs of the anti-MPO variety have beendemonstrated.19 The anti-MPO antibodies reach apeak titer coincident with peak tissue injury.19 His-tological analysis shows that the cellular infiltratein involved tissues is predominantly neutrophilic.17

Mercuric-chloride-induced experimental vasculitisis not entirely similar to the analogous human dis-ease, as is often the case with animal models. Inparticular, necrotizing glomerulonephritis is notseen, although there is membranous glomerulone-phritis (reviewed in Ref. 20). However, although thefactors initiating or predisposing to vasculitis may bequite distinct, and there are differences in the distri-bution of tissue injury, we consider that there aresufficient analogies to permit use of the BN model foranalysis of cellular and humoral elements involved inthe pathogenesis of vasculitis and to examine ther-apeutic interventions.21'22

To examine the role of neutrophils in the patho-genesis of HgCI2-induced vasculitis we have used amonoclonal antibody, RP3, which is a monoclonal ratIgM directed against unknown epitope(s) on rat neu-trophils.23 It was raised against WKA/Hok rat neutro-phils and has been shown to deplete neutrophils in anumber of rat strains.24 Depletion of neutrophils inour model would be predicted to ameliorate vascu-litis. Before examining RP3 in the experimentalmodel in vivo we studied its binding to BN rat neu-trophils and its effect on glycogen-induced influx ofneutrophils into the peritoneum as a way of confirm-ing its capacity to interfere with neutrophil function inthe BN rat.

Materials and Methods

AnimalsBN rats were obtained from our own breeding col-ony, maintained by brother-to-sister mating, andused in age- and sex-matched groups.

Antibody Production and PurificationRP3 was grown in ascites 10 days after pristanepriming of nude mice. It was purified by precipitationwith ammonium sulfate and extensively dialyzingagainst phosphate-buffered saline (PBS) to a con-centration of 0.85 mg/ml.AFRC MAC6 (MAC6) was used as a control anti-

body. It is a rat IgM directed against an irrelevantantigen. The cell line (number 85102268, obtainedfrom the European Collection of Animal Cell Cul-tures) was grown in tissue culture and the antibodywas purified from the supernatant by ammoniumsulfate precipitation and dialysis against PBS. Finalconcentration was 0.89 mg/ml.

BN Neutrophil Staining with RP3 and MAC6Neutrophils were purified on a methyl cellulose gra-dient from 20 ml of heparinized blood obtained fromfour BN rats by cardiac puncture under ether anes-thesia. Cells were incubated for 30 minutes on ice inPBS/1% bovine serum albumin (BSA)/0.1% azidewith the RP3 and MAC6 at a dilution of 1:50 (5 x 105cells in each sample), and washed twice in ice-coldbuffer. The primary antibodies were detected using a1:800 dilution of murine IgG anti-rat IgM antibody(Sigma, Poole, UK) that, after washing, was incu-bated with fluorescein-isothiocyanate-conjugatedanti-mouse IgG (Sigma) at a dilution of 1:1000.

Samples were analyzed using a Becton DickinsonFACSCAN and Lysis 11 software.

Functional Effect of RP3 on OysterGlycogen-Induced PeritonitisA single intraperitoneal injection of 20 ml of oysterglycogen (Sigma) 0.1% in PBS causes significantneutrophil influx into the peritoneum 4 hours later.RP3, MAC6, or PBS was given intravenously at adose of 1 ml at the same time and 2 hours after theglycogen injection. Peritoneal washout was per-formed at 4 hours with 20 ml of PBS injected and 5 mlwithdrawn. The number of neutrophils per milliliter ofwashout was counted using a hemocytometer by anobserver unaware of treatment groups. Results are

Neutrophils in Experimental Vasculitis 83AJPJuly 1996, Vol. 149, No. 1

given as mean neutrophil count per milliliter of wash-out + standard error of the mean (SEM).

Mercuric Chloride Treatment: BasicProtocolAnimals were given HgCI2 (BDH Chemicals, Poole,UK) as a 0.1% solution in distilled water by fivesubcutaneous injections over a 10-day period. Eachinjection consisted of 1 mg/kg body weight. Mono-clonal antibodies were given as daily intravenousinjections in PBS at the doses indicated. Serial bloodsamples were taken from a tail vein under etheranesthesia. Animals were killed at day 5 or day 15after the start of HgCl2 by craniocervical dislocationunder ether anesthesia. Immediate necropsies wereperformed, and the macroscopic gut appearancewas scored by an observer who was unaware of thetreatment groups, using the previously documentedscoring system.22 Tissues were snap-frozen in liquidnitrogen and fixed in formalin for subsequent histo-logical examination.Assessment of tissue injury in the histological sec-

tions was performed by an experienced histopathol-ogist (S. Thiru) without knowledge of the treatmentgroups according to the previously describedscore.22 Large and small bowel were scored sepa-rately, and results are expressed as the sum of thesescores for each animal. Neutrophil infiltrate wasscored as 0, absent; 1, mild; 2, moderate; and 3,marked.

Experimental Design

The therapeutic effect of RP3 on tissue injury at peakdisease was examined in four experiments in whichantibody injections were given on days 10 to 14inclusive. RP3 was used at daily doses ranging from0.25 ml/100 g to 1 ml/100 g. Control animals re-ceived either HgC02 alone or HgCl2 and MAC6 at theappropriate dose. Animals were killed on day 15.

The effect of RP3 on the induction of early tissueinjury was examined in one experiment in whichanimals were given HgCI2 injections on day 1 and 3,daily antibody injections on days 1 to 4, and killed onday 5. Control BN rats received HgCI2 alone orHgCI2 plus MAC6.

Anti-MPO Enzyme-Linked ImmunosorbentAssaySera were assayed for anti-MPO antibodies using asolid phase enzyme-linked immunosorbent assay aspreviously described.19 Briefly, MPO (Calbiochem,

Nottingham, UK) at 5 ,ug/ml in borate buffer wascoated onto microtiter plates (Dynatech, Chantilly,VA) overnight at 40C. After washing, the plates wereincubated with PBS/2% BSA, followed by test orcontrol sera. Bound antibody was detected withalkaline-phosphatase-conjugated rabbit anti-rat an-tibody (Sigma) and substrate (p-nitrophenyl phos-phate; Sigma). Serum from each animal was as-sayed in duplicate with antigen-free wells to controlfor nonspecific binding for each sample. For eachtreatment group results are given as the mean spe-cific binding (binding to antigen-free wells sub-tracted) expressed as a percentage of the specificbinding of a known positive sample included oneach plate.

IgE Enzyme-Linked Immunosorbent AssayWells were coated with MARE-1 (a monoclonal anti-rat IgE heavy chain; Serotec, Oxford, UK) at a con-centration of 5 jig/ml in carbonate buffer at pH 9.6(overnight at 40C) and washed with PBS/1% Tween,and remaining sites were blocked with 5% skimmedmilk in PBS (1 hour at 370C). Sera at a dilution of1 :10,000 in PBS/1% BSA/0. 1% azide were incubatedin duplicate (at 370C for 1 hour); antigen-free wellswere included to control for any residual nonspecificbinding. A solution of rat IgE at 1 ,tg/ml was used asa positive control. After additional washings, boundantigen was detected by an alkaline-phosphatase-conjugated monoclonal anti-rat K and A light chainantibody (Sigma) and substrate, p-nitrophenyl phos-phate (Sigma). Results are expressed as a percent-age of the positive control.

Statistical AnalysisThe Mann-Whitney test was used to analyze differ-ences in macroscopic or histological tissue injuryscore between antibody-treated and control groupsin each experiment. The Kruskall-Wallis test wasused to analyze experiments in which different dosesof RP3 were being compared. Differences in periph-eral neutrophil count at day 14 were analyzed usinga Student's t-test.

Differences were considered significant if the Pvalue was less than 0.05 using a two-tailed test.

Results

Flow CytometryInitial staining of neutrophils with RP3 confirmed thatthis monoclonal antibody binds to neutrophils in the

84 Qasim et alA/P july 1996, Vol. 149, No. 1

10 -

4._0

,C0E

I_ x

0

Q._

0C0

Figure 1. Histogram showing fluorescence distribution of neutrophilsstained w'ith RP3 or MAC6 (fluorescence intensity onz the abscissa,number qf cells on the ordinate). The primnarv laver u'as incubatedwith a mnurine anti-rat IgM second laVer, which was thenl detected uithan aniti-motuse-lgG-fluorescein-isothiocvanate-co nugated an1tibod .RP3 binds to neutrophils, but MAC6 does niot.

BN rat. Figure 1 shows staining of neutrophils withRP3 versus staining with MAC6.

Effect of RP3 on Oyster Glycogen-InducedPeritonitisRP3 significantly reduced glycogen-induced neutro-phil influx into the peritoneum (P = 0.02; Figure 2).There was a small, insignificant reduction of neutro-phil infiltrate with MAC6.

8-

6 -

4 -

2 -

O -

F

0 2 4 6 8 10 12 14

Days after start of HgCI2Figure 3. Peripheral blood neutrophil count in controls given HgCl,alone or RP3 on days 10 to 14 after HgCl2 (showing mean countt n =5 and error bars shooing SEM). At day 14, RP,3-treated animals hada significantlv reduced peripheral neutrophil count (P < 0.05).

Effect of RP3 in Vivo

HgCI2 induced a 2- to 10-fold rise in blood neutrophilnumbers by day 15. At 1 ml/100 g, RP3 this rise wasvirtually abolished, indicating depletion of peripheralneutrophils (Figure 3). The difference in neutrophilcount on day 14 between RP3-treated and controlanimals was statistically significant (P = 0.009).

At a dose of 1 ml/100 g given on days 10 to 14,RP3 significantly ameliorated tissue injury (P = 0.03)whereas 1 ml/100 g MAC6 had no protective effect(Table 1). Doses of 0.5 and 0.25 ml/100 g caused asmaller reduction in tissue injury, although the latterdid not reach statistical significance (Table 1, mac-roscopic score, Kruskall-Wallis P = 0.08). The histo-logical score was similar (Table 2, Kruskall-WallisP = 0.06) with a striking reduction in the degree ofneutrophil infiltrate with the higher dose of RP3(Kruskall-Wallis P = 0.05). Figure 4 shows sectionsof large bowel on day 5 (grades 1 and 2 tissue injury)

Table 1. Macroscopic Gut Scoreb

0

20 -x

o 15 -0-0

0.

a-

o -

Controls MAC 6 RP3

Figure 2. The effect ofRP3 and MAC6 on- glvcogen-induced iunflux ofneutrophils into the peritoneum. MAC6 caused a nionsignificanit re-duction in neutrophil inflox, but RP,3 caused a greater, significanteffect. Bars shou mean score (n = 6) and error bars show SEM.

Treatment(daily dose/1OOg)

Day 15HgCI2MAC6 (1 ml)MAC6 (0.5 ml)RP3 (1 ml)RP3 (0.5 ml)RP3 (0.25 ml)

Day 5HgCl2MAC6 (0.5 ml)RP3 (0.5 ml)

Mean external gut score(SEM)

2.8 (0.3)2.3 (0.3)2.7 (0.7)0.5 (0.5)1 (0.4)1.8 (0.5)

1.75 (0.90)0.75 (1.2)0 (0)

Mean score (on day 15 or day 5 as indicated) and SEM areshown (n = 3 to 9). Reduction of tissue injury by RP3 is dosedependent, being greatest at a dose of 1 ml/100 g. MAC6 had nosignificant effect on tissue injury.

+ A_.;L- --I.. :_ :_

II r,

v -

Neutrophils in Experimental Vasculitis 85AJPJuly 1996, Vol. 149, No. 1

Table 2. Histolgical anid Neu:trophil Tissue Injury Scores

Treatment group(daily dose/100 g)

HgCI2MAC6 (0.5 ml)RP3 (0.5 ml)RP3 (0.25 ml)

Mean tissueinjury score

(SEM)

7.5 (0.5)6.3 (0.9)3.8 (0.7)6.0 (0.9)

Meanneutrophil

score (SEM)

4 (0)4 (0)0.5 (0.5)2.25 (0.6)

Scores were from day 15 in animals treated with 0.5 and 0.25ml/100 g/day on days 10 to 14. Mean score -+ SEM are shown(n = 5). RP3 reduces the tissue injury and neutrophil scoresalthough only the effect of 0.25 ml is significant.

and day 15 (grades 3 and 4 vasculitis with fibrinoidnecrosis and a dense neutrophil infiltrate) afterHgCI2. The section of bowel from an animal treatedwith 1 ml RP3/100 g is virtually normal, with minimaledema in the submucosa and no cellular infiltrate.

In the experiment to examine the effect of RP3 on

the induction of early tissue injury, two out of fiveanimals given HgCI2 alone, one out of four givenHgCI2 with MAC6, and one of four given RP3 hadmacroscopic evidence of tissue injury (Table 1shows the mean scores in each group). On histolog-ical assessment, these results were confirmed ex-

cept that one of the RP3-treated animals had histo-logical evidence of vasculitis. The differences were

not statistically significant.

Anti-MPO and IgE Levels

There was no difference in anti-MPO or IgE levelsbetween RP3-treated animals and controls (Figure 5shows anti-MPO levels, data not shown for IgE). Allgroups showed the expected rise in anti-MPO andIgE levels.

DiscussionIn this study we have shown for the first time in vivothat neutrophils play a key role in the pathogenesis ofvasculitis by demonstrating that vasculitis can beameliorated by depletion of neutrophils. The mono-

clonal antibody RP3 was shown to bind to BN ratneutrophils and to reduce oyster glycogen-inducedinflux of neutrophils into the peritoneum. Treatmentwith the control antibody MAC6 was associated witha small reduction in glycogen-induced peritonitis,but this was not statistically significant. This con-

firmed that RP3 was capable of functional effects inthe BN rat. When tested in vivo, RP3 caused a dose-dependent amelioration in tissue injury when givenon days 10 to 14. This was associated with a reduc-tion in the degree of tissue neutrophil infiltrate and an

abolition of the HgCI2-induced rise in peripheral neu-trophil count. When given during the induction ofvasculitis, RP3 treatment was again associated witha reduction in tissue injury, but this did not reachstatistical significance, partly because in this exper-iment tissue injury in controls was unexpectedly mild.Vasculitis is usually seen in 95% of animals by day 5after HgCI2, with a mean tissue injury score of 2.6(own observations and previously published data).17Many factors have been implicated in the etiology

and pathogenesis of the primary vasculitides in man.Early studies suggested a genetic predisposition asthere appeared to be a link between certain vascu-litic diseases and major histocompatibility loci, eg,HLADRw2 with Goodpasture's disease and HLAB8with Wegener's granulomatosis, but the largest studyto date does not demonstrate any clear-cut associ-ations between alleles at the major histocompatibilityloci DR, DQ, or DP and systemic vasculitis.25-27Circumstantial evidence supports a pathogenic rolefor infection; certain infective agents are stronglyassociated with vasculitic diseases, eg, hepatitis Bwith polyarteritis nodosa, intercurrent infections mayprecipitate disease relapse, and antibiotics are re-ported to promote or maintain disease remis-sion.82829 The mechanism of endothelial damagerelated to infections could be direct (by complementactivation or by generation of immune complexesthat invoke an inflammatory response when depos-ited in blood vessels) or permissive (the response toan infective organism creates a cytokine environ-ment that primes leukocytes so that any further acti-vation, for example, by ANCA of neutrophils, resultsin vasculitic damage). In addition to ANCA, autoan-tibodies against endothelial cells may also be de-tected in primary vasculitides.0'31 It is unproven thatthese antibodies are pathogenic in vivo, but ANCAshave been shown to activate primed neutrophils invitro and autoantibodies against endothelial cells tocause complement-mediated lysis of cultured endo-thelial cells.7'32'33 In vivo evidence for damage to theendothelium by activated leukocytes is mostly indi-rect, consisting mainly of histological data showing Tcells and/or neutrophils in vasculitic lesions.","3The mechanism of initiation of vasculitis in the

HgCI2 model is unclear. There is evidence that theautoimmune syndrome is T cell dependent,15 andindirect support for the hypothesis that it is driven bythe Th2 cell subset of helper CD4+ T cells,36 but adirect role for T cells in the pathogenesis of thevasculitis is unproven. The rapid onset of tissue in-jury, which can occur within 24 hours of the firstinjection of HgCl2, suggests that cells other than Tcells are involved in the initiation of vasculitic injury.

86 Qasim et alAJPjulv 1996, Vol. 149, No. 1

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Neutrophils in Experimental Vasculitis 87A/PJuly 1996, Vol. 149, No. 1

120 -

100 -

EControls0 80 - ORP3> =ziantibody treatmentU)o 60-

40 -

401

20

5 10 15

Days after start of HgCI2

Figure 5. Aniti-MPO levels (expressed aspercentage ofpositive conltrol)in animals given I mnl RP3 on days 10 to 14 compared uith controlsgiven HgCL, alone. The mean titer at each tinepoint ( n = 5) and SEMare shown. RP3 treatment had nlo significant effect on anti-MPO titer.

We have recently proposed that mast cells, whichproduce and release a number of potent neutrophilchemoattractants including tumor necrosis factor37and IL-838 and which are found in large numbers inthe gut, may be important in this early phase ofHgCI2-induced tissue injury and may also play a rolein driving the Th2 autoimmune response by produc-ing IL-4. Our evidence demonstrates that sub-stances that induce autoimmunity in the BN rat canactivate its mast cells and increase the expression ofmRNA for IL-4.39 It may also be possible that HgCI2itself can directly activate neutrophils, although thiswould not explain the distribution of tissue injury.

The amelioration of vasculitis by an antibody thatdepletes neutrophils is one of the first pieces ofdirect evidence that neutrophils play a role in thepathogenesis of vasculitis. RP3 has previously beenused in vivo to examine the role of neutrophils insubcutaneous inflammatory responses occurringover time periods of up to 24 hours. Neutrophil de-pletion using RP3 caused a reduction in tumor-necrosis-factor-induced vascular hyperpermeabil-ity24 and IL-8-induced CD4+ recruitment intosubcutaneous tissue,40 providing evidence that neu-trophils can damage blood vessels and affect themigration of lymphocytes. The reduction in vasculitisby RP3 in our study suggests that neutrophils, at-

tracted by release of chemoattractants from mastcells, lymphocytes, or endothelium, can cause sig-nificant tissue injury. It is still not clear, however,whether they do this directly by release of degrada-tive enzymes or by recruitment of other cells such aslymphocytes or macrophages. It is unlikely that re-active oxygen species are sufficient to cause tissueinjury as an earlier study has shown that antioxidantsare not protective.21

Despite the effects of RP3 on tissue injury, therewas no significant effect on the level of anti-MPOantibodies or IgE titer. The latter indicates that themonoclonal antibodies have not interfered with thepolyclonal autoimmune response and, by inference,not affected T or B cells. There could be a number ofexplanations for the former. For example, perhapsanti-MPO antibodies are not of pathogenic impor-tance, at least in this model, or that anti-MPO anti-bodies may be pathogenic but that through actual orfunctional depletion of neutrophils they have losttheir substrate.

The data presented here confirm that neutrophilsplay an important role in the pathogenesis of tissueinjury in HgCI2-induced experimental vasculitis. Theprotective effect of RP3 at peak disease clearly hasimplications for the use of a neutrophil-depleting an-tibody in the treatment of disease in man. This studyconfirms that HgCI2-induced experimental vasculitisprovides a useful model for the study of therapeuticinterventions and the pathogenesis of vasculitis.

Acknowledgments

We thank Margaret MacLeish for the preparation ofsamples for histological analysis and Karen Wolfreysand Barbara Sgotto for help with antibody purifica-tion.

References

1. Hunder GG, Arend WP, Bloch DA, Calabrese LH, FauciAS, Fries JF, Leavitt RY, Lie JT, Lightfoot RW, Masi AT,McShane DJ, Michel BA, Mills JA, Stevens MB, WallaceSL, Zvaifler NJ: The American College of Rheumatol-

Figure 4. Sections oflarge bowel showing effect ofRP3 oni HgCGL-induced vasculitis. A: Nonnal large bowel with closely packed tubular glands in themnucosa, a thin layer ofsubmzucosa composed offibrovascular tissue, and uniremnarkable muscle layer. H&E,; magnification, X 180. B: Grades 1 aud2 tissue injury otn dacy 5. Marked widening of the submucosa secontdary to edemna and vascular dilatation. There is an- early intflammatory cellinfiltrate at the base ofthe glands in the mucosa and in the submucosa u'ith perivascular accentuation. H&E; magnification, X 120. C: Grades 3 and4 vasculitic lesions on day 15. There isfibrinoid necrosis of smnall arteries uith an initense neutrophil infiltrate surrountdintg niecrotic vessels. Somnevessel segments (A) shou' totalfibrinoid niecrosis uhereas elsewhere there is infiltratiotn wvith nteutrophils butpreservation ofvessel architectulre (B) Notethat the intfammatory intfiltrate extends to the muiscle layer ( ie, tissue intjury involves the full thickness ofthe vessel wall). H&E; mnagnification, X 120.D: Section of bowel ont day 15 from ant animnal treated with RP3 I ml/100 gday on davs 10 to 14. There is no evidence of vasculitic levsions, ntonieutrophil infiltrate, anid minimal suibmnucosal edemna. H&E, magnification, X 180.

88 Qasim et alAJPJuly 1996, Vol. 149, No. I

ogy 1990 criteria for the classification of vasculitis.Arthritis Rheum 1990, 33:1065-1067

2. Lockwood CM, Jones SJ, Jayne DRW, Lai KN: ANCAand systemic vasculitis. Netherlands J Med 1990, 36:154-156

3. Gupta SK, Niles JL, McCluskey RT, Arnaout MA: Iden-tity of Wegener's autoantigen (p29) with proteinase 3and myeloblastin. Blood 1990, 76:2162

4. Cohen Tervaert JW, Goldschmeding R, Van der HemGK, Hene RJ, Koolen Ml, von den Borne AEGK, Kal-lenberg CGM: Autoantibodies to myeloperoxidase areassociated with different forms of vasculitis. ArthritisRheum 1990, 33:1264-1272

5. Jennette JC, Wilkman AS, Falk RJ: Anti-neutrophil cy-toplasmic autoantibody-associated glomerulonephritisand vasculitis. Am J Pathol 1989, 135:921-930

6. Ewert BH, Jennette JC, Falk RJ: Anti-myeloperoxidaseantibodies stimulate neutrophils to damage human en-dothelial cells. Kidney Int 1992, 41:375-383

7. Keogan MT, Fujimoto T, Brown DL, Lockwood CM:Activation of neutrophils by anti-neutrophil cytoplasmantibodies. Q J Med 1991, 80:789

8. Pinching AJ, Rees AJ, Pussell BA, Lockwood CM,Mitchison RS, Peters DK: Relapses in Wegener'sgranulomatosis: the role of infection. Br Med J 1980,281:836-838

9. Deremee RA: The treatment of Wegener's granuloma-tosis with trimethoprim/sulfamethoxazole: illusion or vi-sion? Arthritis Rheum 1989, 31:1068-1073

10. Savage COS, Lockwood CM: Systemic vasculitis. Clin-ical Aspects of Immunology, vol. 2. Edited by PJ Lach-mann, DK Peters, FS Rosen, MJ Walport. Boston,Blackwell Scientific Publications, 1993, pp 1205-1215

11. Lehrer RI, Ganz T, Selsted ME, Babior BM, Curnutte JT:Neutrophils and host defence. Ann Intern Med 1988,109:127-142

12. Malech HD, Gallin JI: Neutrophils in human diseases.N Engl J Med 1988, 317:687-692

13. Cochrane CG: Immunological tissue injury mediatedby neutrophil leukocytes. Adv Immunol 1968, 9:99-108

14. Henson PM: Pathological mechanisms in neutrophilmediated injury. Am J Pathol 1972, 68:593-612

15. Pelletier L, Pasquier R, Vial M-C, Mandet C, Moutier R,Salomon JC, Druet P: Mercury-induced autoimmuneglomerulonephritis: requirement for T cells. NephrolDial Transplant 1987, 1:211-218

16. Mathieson PW, Thiru S, Oliveira DBG: Mercuric chlo-ride-treated Brown Norway rats develop widespreadtissue injury including necrotizing vasculitis. Lab Invest1992, 67:121-129

17. Qasim FJ, Mathieson PW, Thiru S, Oliveira DBG: Timecourse and characterization of mercuric chloride in-duced autoimmunity in the Brown Norway rat. J Auto-immun 1995, 8:193-208

18. Mathieson PW, Thiru S, Oliveira DBG: Regulatory roleof OX22+ T cells in mercury-induced autoimmunity inthe Brown Norway rat. J Exp Med 1993, 177:1309-1316

19. Esnault VLM, Mathieson PW, Thiru S, Oliveira DBG,

Lockwood CM: Autoantibodies to myeloperoxidase inBrown Norway rats treated with mercuric chloride. LabInvest 1992, 67:114-120

20. Mathieson PW, Qasim FJ, Esnault VLM, Oliveira DBG:Animal models of systemic vasculitis. J Autoimmun1993, 6:251-264

21. Qasim FJ, Mathieson PW, Thiru S, Oliveira DBG: Effectof steroids and antioxidants on experimental vasculitis.Clin Exp Immunol 1994, 98:66-70

22. Qasim FJ, Mathieson PW, Thiru S, Oliveira DBG: Cy-closporin A exacerbates mercuric chloride-inducedvasculitis in the Brown Norway rat. Lab Invest 1995,72:183-190

23. Skiya S, Gotoh S, Yamashita T, Watanabe T, Saitoh S,Sendo F: Selective depletion of rat neutrophils by in vivoadministration of a monoclonal antibody. J LeukocyteBiol 1989, 46:96-102

24. Abe Y, Sekiya S, Yamasita T, Sendo F: Vascular hyper-permeability induced by tumor necrosis factor and itsaugmentation by IL-1 and IFN--y is inhibited by selec-tive depletion of neutrophils with a monoclonal anti-body. J Immunol 1990, 145:2902-2907

25. Rees AJ, Peters DK, Compston DAS, Batchelor JR:Strong association between HLA DRw2 and antibodymediated Goodpasture's syndrome. Lancet 1978,i:966-968

26. Katz P, Alling DW, Haynes BF, Fauci AS: Association ofWegener's granulomatosis with HLA-B8. Clin ImmunolImmunopathol 1979, 14:268-272

27. Zhang L, Jayne DRW, Zhao MH, Lockwood CM, Ol-iveira DBG: Distribution of MHC class 11 alleles in pri-mary systemic vasculitis. Kidney Int 1995, 47:294-298

28. Guillevin L, Lhote F, Sauvaget F, Deblois P, Rossi F,Levallois D, Pourrat J, Christoforov B, Trepo C: Treat-ment of polyarteritis nodosa related to hepatitis B viruswith interferon-a and plasma exchanges. Ann RheumDis 1994, 53:334-337

29. Deremee RA, McDonald TJ, Weiland LH: Wegener'sgranulomatosis: observations on treatment with anti-microbial agents. Mayo Clin Proc 1985, 60:27-32

30. Ferraro G, Meroni PL, Tincani A, Sinico A, Barcellini W,Radice A, Gregorini G, Froldi M, Borghi MO, BalestrierrG: Anti-endothelial cell antibodies in patients with We-gener's granulomatosis and micropolyarteritis. ClinExp Immunol 1990, 79:47-53

31. Frampton G, Jayne DRW, Lockwood CM, Cameron JS:Autoantibodies to endothelial cells and neutrophil cy-toplasmic antigens in systemic vasculitis. Clin Exp Im-munol 1990, 82:227-232

32. Ewert B, Falk RJ, Jenette JC: Anti-neutrophil cytoplas-mic antibodies stimulate neutrophils to injure endothe-lial monolayers in vitro. Am J Kidney Dis 1991, 18:28

33. Savage COS, Pottinger BE, Gaskin G, Lockwood CM,Pusey CD, Pearson J: Vascular damage in Wegener'sgranulomatosis and microscopic polyarteritis: pres-ence of anti-endothelial cell antibodies and their rela-tion to anti-neutrophil cytoplasm antibodies. Clin ExpImmunol 1991, 85:14-19

Neutrophils in Experimental Vasculitis 89AJPju4l 1996, Vol. 149, No. 1

34. Hooke DH, Hancock WW, Gee DC, Kraft N, Atkins RC:Monoclonal antibody analysis of glomerular hypercel-lularity in human glomerulonephritis. Clin Nephrol1984, 22:163-168

35. Savage COS, Winearls CJ, Evans DJ, Rees AJ, Lock-wood CM: Microscopic polyarteritis: presentation, pa-thology and prognosis. Q J Med 1985, 56:467-483

36. Prouvost-Danon A, Abadie A, Sapin C, Bazin H, DruetP: Induction of IgE synthesis and potentiation of anti-ovalbumin IgE antibody response by HgCI2 in the rat.J Immunol 1981, 126:699-702

37. Zhang Y, Ramos BF, Jakschik BA: Neutrophil recruitmentby tumor necrosis factor from mast cells in immune com-plex peritonitis. Science 1992, 258:1957-1959

38. Moller A, Lippert U, Lessmann D, Kolde G, Hamann K,Welker P, Schadendorf D, Rosenbach T, Luger T,Czarnetzki BM: Human mast cells produce IL-8. J Im-munol 1993, 151 :3261-3266

39. Oliveira DBG, Gillespie K, Wolfreys K, Mathieson PW,Qasim F, Coleman JW: Compounds that induce auto-immunity in the Brown Norway rat sensitize mast cellsfor mediator release and interleukin-4 expression. EurJ Immunol 1995, 8:2259-2264

40. Kudo C, Araki A, Matsushima K, Sendo F: Inhibition ofIL-8 induced W3/25 (CD4+) T lymphocyte recruitmentinto subcutaneous tissues of rats by selective depletionof in vivo neutrophils with a monoclonal antibody. J Im-munol 1991, 147:2196-2201