different pathways leading to cutaneous leukocytoclastic vasculitis in mice

Exp Dermatol 2001: 10: 391–404 Copyright C Munksgaard 2001Printed in Denmark ¡ All rights reserved EXPERIMENTAL DERMATOLOGY

ISSN 0906-6705

Different pathways leading to cutaneousleukocytoclastic vasculitis in mice

Sunderkotter C, Seeliger S, Schonlau F, Roth J, Hallmann R, Luger TA, C. Sunderkötter1,2, S. Seeliger2,Sorg C, Kolde G. Different pathways leading to cutaneous leukocytoclastic F. Schönlau2, J. Roth2,vasculitis in mice. R. Hallmann4, T. A. Luger1,Exp Dermatol 2001: 10: 391–404. C Munksgaard, 2001 C. Sorg2 and G. Kolde3

1Department of Dermatology, University ofAbstract: To investigate the pathomechanisms of leukocytoclastic vas-Münster, Münster, Germany; 2Institute ofculitis (LcV) we compared mouse models of LcV with non-vasculitic irri- Experimental Dermatology, University of

tant contact dermatitis (ICD). Criteria for LcV as met by the immune Münster, Münster, Germany; 3current address:complex-mediated Arthus reaction (Art-r) were also fulfilled by the lo- Department of Dermatology, Humboldtcalized Shwartzman reaction (Shw-r) and by cutaneous Loxoscelism (Lox) University of Berlin, Berlin, Germany;(injection of venom from Loxosceles reclusa containing sphingo- 4Department Experimental Medicine I, University

of Erlangen, Erlangen, Germanymyelinase D). After depletion of PMN (by g-irradiation) vessel damagecould not be elicited in these models, distinguishing them from modelsof direct endothelial insult (necrotizing ICD). Depletion of complementcould only delay, but not inhibit the Art-r, and did not change ICD, Loxor the Shw-r. The Shw-r exclusively revealed a sustained local expressionof vascular adhesion molecules for 24 h in the preparatory phase (LPSs.c.), not observed in the Art-r, in Lox or ICD. Subsequent challenge withLPS i.p. was associated with upregulation of Mac-1 and ICAM-1 on PMN,but not of VLA-4 or LFA-1 (FACS analysis). Cytokines which were ableto replace LPS in priming for LcV in the Shw-r (TNF-a and IL-1b) alsoinduced sustained expression of adhesion molecules, whereas IL-12 andIFN-g did neither. Neutralizing IL-12 or IFN-g also inhibited neither LcVnor sustained expression of adhesion molecules, whereas anti-TNF-a in- Key words: vasculitis – Arthus reaction –hibited both. Anti-TNF-a had no marked inhibitory effects in the Art-r, in Shwartzman reaction – sphingomyelinase –Lox or ICD. Combined (but not separate) neutralization of both E-se- E-selectin – VCAM-1 – complement – diapedesislectin and VCAM-1 by antibodies suppressed LcV independent from reduc- Cord Sunderkötter, Department of Dermatologying influx of PMN, proving that their sustained expression is decisive for and Institute of Experimental Dermatology,the Shw-r and interferes with normal diapedesis. Since Loxosceles venom University of Münster, von Esmarch Str. 56,is known to dysregulate diapedesis and degranulation of PMN in vitro, D-48149 Münster, Germanysince adherent immune complexes activate PMN at the vessel wall, and Tel.: π49 251 8356578

Fax: π49 251 8356549since adhesion molecules are dysregulated in the Shw-r, we suggest thate-mail: sunderk/uni-muenster.deLcV develops when activation of PMN coincides with vascular alter-

ations which interfere with normal diapedesis. Accepted for publication 9 May 2001

Introduction

The term vasculitis denotes an inflammatory con-dition in which destruction of the wall of bloodvessels by leukocytes is the primary event. Leuko-cytoclastic vasculitis is the inflammation of small

Abbreviations:Art-r: Arthus reaction; Shw-r: Shwartzman reaction; Lox: cu-taneous loxoscelism; ICD: irritant contact dermatitis; HSA: hu-man serum albumin; mAb: monoclonal antibody; CVF: cobravenom factor; LcV: leukocytoclastic vasculitis; PMN: polymor-phonuclear granulocytes.

391

blood vessels (usually post-capillary venules) andis characterized histologically by infiltration ofgranulocytes (PMN), karrhyorhexis of their nuclei(leukocytoclasia) and damage of the vessel wall,coupled with compromise of the lumen and ex-travasation of erythrocytes (for review (1, 2)).

The term leukocytoclastic vasculitis is frequentlyused synonymously with the term immune-com-plex-vasculitis, as the precipitation of circulatingimmune complexes along the wall is widely be-lieved to be the predominant cause for the damageof small vessels. However, other inflammatory pro-cesses such as infection of endothelial cells by mi-

Sunderkotter et al.

crobes or the endotoxin-mediated Shwartzman re-action (Shw-r) also have been reported to involvedamage of small blood vessels (for review (1–3)).Similarly, bites from the spider Loxosceles reclusacause local hemorrhagic necrosis, which is sugges-tive of vasculitis (4). The active ingredient here issupposed to be sphingomyelinase D which hasbeen shown to be an activator of endothelial cellswith characteristic differences to LPS or TNF-a(5). It is not exactly known by which pathomech-anisms immune-complexes, endotoxins or venomof Loxosceles reclusa lead to damage of the vascu-lar wall and why some inflammations featuring agranulocytic infiltrate are associated with destruc-tion of blood vessels whereas most other acute gra-nulocytic inflammations are not.

To dissect the pathophysiological events whichare distinct for acute vasculitis, we analyzed inmice different models of inflammation with andwithout vessel damage. By using different elicitingagents we show that histological and clinical cri-teria of LcV, as regularly encountered in the im-mune complex-mediated Arthus reaction (Art-r),are also fulfilled by at least two other inflamma-tory reactions: by the localized LPS-inducedShwartzman reaction (Shw-r) and by cutaneousLoxoscelism (Lox). Thus, comparative studies be-tween these models and irritant contact dermatitisas a non-vasculitic inflammation offer a good ap-proach to characterize pathomechanisms of vas-culitis and to distinguish mechanisms common toall acute inflammations from those specific for vas-culitis. In the Shw-r, but not in the Art-r, Lox orICD, we found a markedly sustained expression ofadhesion molecules on endothelial cells (E-selectinand VCAM-1) and an increase of CD18 on circu-lating leukocytes. Agents shown to elicit or inhibitthe Shw-r also influenced the characteristic expres-sion patterns of adhesion molecules, indicatingtheir relevance for the pathophysiology of vas-culitis.

Materials and methodsAnimal models

Animals: Male BALB/c mice (10–14 weeks of age)were obtained from Charles River, (Sulzberg, Ger-many).

a) Reverse passive Arthus reaction (Art-r): TheArt-r was elicited by injecting rabbit- or goat-anti-human serum albumin (HSA) (20 ml) subcutane-ously (s.c.) (Sigma, Taufkirchen, Germany) in theleft ear and 2.5 mg HSA (Sigma) intravenously(i.v.) (Fig. 1).

b) Localized Shwartzman reaction (Shw-r): Forthe Shw-r 7.5 mg LPS (Escherichia coli serotype055:B5, Sigma) were injected s.c. into the left ear

392

Figure 1. Scheme of the different animal models for LcV andnon-vasculitic ICD, indicating relevant time intervals.

(preparatory dose). For challenge, i.e. for elici-tation of a vasculitic reaction, 150 mg LPS wereinjected intraperitoneally (i.p.) 24 h later (Fig. 1).

c) Cutaneous Loxoscelism (Lox) was induced bys.c. injection of 0.2 ml spider venom (lyophilisatepurchased from Spider Pharm, Feasterville, PE,USA) dissolved in normal saline into the left ear.The active ingredient is sphingomyelinase D,which, in contrast to sphingomyelinase C convertssphingomyelin in ceramide-P and not ceramide.Since sphingomyelinase C (obtained from Staphaureus) (Sigma) (2.5 mg dissolved in PBS) is com-mercially available in a standardized form, it wasalso injected to see if it would cause a similar reac-tion as Loxosceles venom (Fig. 1).

d) Irritant contact dermatitis (ICD): ICD wasinduced by applying 2.5%, necrotizing ICD by ap-plying 50% benzalkonium chloride (Sigma) dis-solved in olive oil: acetone (1:5 v/v) on the dorsalaspect of the ear (Fig. 1).

All animal experiments were approved by thestate review board (Münster, G 24/96, 46/93, 38/90).

Macroscopic criteria for evaluating severity ofinflammation and vessel damage were i) ear thick-ness as a parameter for edema and infiltrate inICD, and ii) petechiae or hemorrhage for LcV. Pe-techiae or hemorrhage were measured semi quanti-tatively using a score from 1 to 4:

1 Ω1–10 min petechiae (‘‘size of the tip of a pin’’)or 5 small petechiae (‘‘head of a pin’’);

2 Ω5–15 small petechiae, or hemorrhagic macula(up to 0.5 mm diameter);

3 Ωhemorrhagic maculae between .0.5 mm and,1.0 mm, or 2 hemorrhagic maculae of ,0.5mm;

4 Ωhemorrhagic area covering 50% of the ear’ssurface.

Pathways in leukocytoclastic vasculitis

In order to quantify vessel damage we alsomeasured the amount of extravasated FITC-labeled bovine serum albumin, in modification tothe use of radiolabeled albumin (6) or Evans bluedye bound to albumin (7) in other studies. Micewere injected i.p. with 2.5 mg FITC-labeled BSA(Sigma) in 200 ml PBS at elicitation of the inflam-matory reaction. Skin punches of 6 mm were har-vested from the ears after 6 h, cut in slices of 1mm and subjected to 2 ml trypsin for 40 min. Theextravasated fluorescent dye was measured using afluorescence photometer at 517 nm (FluorMax2with DataMax for WindowsTM) (ISA, Jobin Yvon-Spex, Instruments S.A., Inc. New Jersey) (8).

For depletion of granulocytes whole body ir-radiation was performed at a dose of 6 Gy, util-izing 60Co at an approximate dose rate of 1.45 Gy/min. Experiments were started 8–9 days after ir-radiation when peripheral blood count yielded ,5leukocytes/mm3.

For depletion of complement, mice were injectedi.p. with Cobra venom factor (CVF) (Sigma) 14 mg(3¿2.1 U) of CVF 16 h, 8 h and 1 h before elicitingthe Art-r, the Shw-r, Lox or ICD. It contains cobraC3b which forms with murine Bb a non-degrad-able C3 convertase (C3bBb) depleting murinecomplement by continuous activation. Comple-ment levels in serum were kindly determined by thelaboratory of Prof. Ringels (Aachen, Germany).No hemolytic activity was found within 48 h aftertreatment with CVF. CVF (14 mg) was given i.p.also for activation of complement in mice not pre-viously depleted of complement.

Cytokines

Recombinant murine IL-1b, IL-12, IFN-g, andTNF-a as well as the corresponding neutralizingantibodies were obtained from Genzyme (Rüssels-heim, Germany). All injected solutions (except forLPS) were found to contain less than 0.05–0.1 en-dotoxin units per ml when tested in the Limulusamebocyte lysate assay (E-Toxate from Sigma).

Processing of tissue samples

For histological analysis 6 to 10 mice of each strainwere sacrificed at designated time points (for thekinetic studies in each model after 1 h, 3 h, 6 h, 8h, 12 h, 24 h, 48 h, 72 h; in the Shwartzman reac-tion also 1 h, 2 h, 4 h, and 6 h after LPS i.p.).In those experiments in which CVF, cytokines orneutralizing antibodies were injected or where micewere depleted of leukocytes, only characteristictime points (e.g. 3 h, 6 h, 24 h after LPS i.p.) werechosen for examination of tissue. Ears were excisedand cut longitudinally in two halves for prepara-

393

tion of paraffin and cryostat sections. Cryostat sec-tions were stained: i) for immunofluorescence withgoat anti murine C3 conjugated with FITC (Orga-non Teknika/Cappel, Eppelheim, Germany) and ii)for immunohistochemistry using the indirect im-munoperoxidase assay as described in detail pre-viously (9). The following antibodies were used:mAb 10E9.6 (rat IgG2a) against murine E-selectin(kindly provided by D. Vestweber, Münster, Ger-many (10)); mAb M/K-1.9 (rat IgG1) against mu-rine VCAM-1 (ATCC, Rockville, MD, USA) (11);mAb YN1/1.7.4 (IgG2b) against murine ICAM-1 (kindly provided by F. Takei, Vancouver, B.C.,Canada); mAb M1/70 (rat IgG2b) against Mac-1(granulocytes and macrophages); mAb RB6–8C5(rat IgG2b) against Gr-1 (Ly-6G) (granulocytes);mAb 53–7.3 (rat IgG2a) against Lyt-1 on T cellsand B-cell subsets (all by Pharmingen, Hamburg,Germany); mAb PS2 (rat IgG2b) against VLA-4;mAb F4/80 (rat IgG2b) for murine macrophages(all by Serotec, Wiesbaden, Germany); goatF(abø)2 anti-rat IgG and goat F(abø)2 anti-rabbitIgG, conjugated with peroxidase (Dianova, Ham-burg, Germany). Antibodies and, for control, iso-types of (irrelevant) IgG were diluted in PBS with1% BSA (maximal concentration 1.5 mg IgG/ml).

Microscopic evaluation

For counting of positive cells 160-fold magnifi-cation was used in association with an ocular en-dowed with an eyepiece graticule. To compareareas of equal size 10 graticule fields were evalu-ated in each section. Results were presented as ab-solute number of positive endothelial cells (E-se-lectin, VCAM-1) in the defined areas or as percen-tage of positive cells (leukocytes) in the infiltrate.

Flow cytometry

Before, and 15 min after systemic application ofHSA, LPS, CVF or a cytokine cocktail containingIFN-g, IL-1b and TNF-a, blood was drawn fromthe axillary region of 10 mice per group and kepton ice. After removal of erythrocytes by osmoticshock, leukocytes were stained with mAb againstMac-1, LFA-1, VLA-4 or ICAM-1 as describedpreviously (9). Flow cytometric analysis was per-formed with a FAC-Scan (Becton Dickinson)using Lysis software. Parameters were percentageof positive cells and mean density, the latter calcu-lated as sum of fluorescence of all cells, divided bynumber of cells and divided by mean from fluor-escence of isotype (control). Gating of PMN wascontrolled by staining with mAb against Gr-1.

Sunderkotter et al.

Figure 2. Extravasation of FITC-BSA as a marker for extravasation from damaged vessels (fluorescence activity of the extravasateddye given in c.p.s. (counts per second) was measured using a fluorescence photometer; data are given as mean∫SD, nΩ6). a) Art-r:After 6 h we observed a significant increase of intensity at 517 nm as a marker of extravasation of FITC-BSA and as a sign ofblood vessel damage, in contrast to the untreated right ear and to mice which received only FITC-BSA (Fig. 2a). b) Shw-r: Six hafter systemic challenge with LPS i.p. the pretreated ear showed a significant increase of intensity in contrast to non-primed sites(Fig. 2b). The intensity was not as high as in the Art-r or in Lox, reflecting the smaller size of hemorrhagic lesions. Unchallengedsites also showed an increase in intensity after the same time interval (i.e. 24 hπ6 h), suggestive of increased vascular permeabilityduring the course of priming (Fig. 2b), but it did not reach the high levels associated with vascular damage. c) Cutaneous loxoscelism(Lox): FITC measurement revealed significant extravasation of FITC-BSA as a sign of vessel damage within 6 h (Fig. 2c). d) ICDincluding necrotizing ICD: ICD revealed some extravasation of FITC-BSA indicative of intense vasodilatation and coinciding withthe intense ear swelling usually observed in ICD with 5% benzalkonium chloride. In ICD with necrotizing concentrations of irritantvessel damage in the course of general tissue destruction lead to values for FITC-BSA similar as observed in the models of primaryvasculitis (Fig. 2d).

Neutralization of E-selectin and VCAM-1

For neutralization of E-selectin and VCAM-1 inthe Shw-r 200 mg of UZ4 (rat IgM) against murineE-selectin (12) and of M/K-1.9 (rat IgG1) (11)against murine VCAM-1 as well as correspondingconcentrations of isotypes (rat IgG1 and IgM),were injected i.v. into 7 mice each just prior to in-jection of LPS in the challenge phase. Hemorrhagewas evaluated over a period of 6 h. In another ex-periment ears from 6 mice each were harvested 2h after LPS i.p. (prior to onset of hemorrhage) fortissue extraction of myeloperoxidase which is anestablished parameter for the amount of granulo-cytes and monocytes in the tissue (13). Tissuesamples were homogenized and sonicated in PBS.MPO activity in supernatants was measured by thechange in optical density (at 460 nm) resultingfrom decomposition of H2O2 in the presence of O-dianisidine (13).

394

Statistical analysis

Results were expressed as arithmetic means(usually nΩ6–10, not ,5) with standard deviation.The U-test according to Mann and Whitney wasperformed to determine significant differences.Values of P,0.05 were considered to be significant.

ResultsClinical picture: hemorrhagic lesions occur in theArthus (Art-r), Shwartzman reaction (Shw-r), andin cutaneous loxoscelism (Lox), but not in ICD

a) Art-r. Within 2 h after injection of anti-HSA s.c.and of HSA i.v. ears showed infiltration and finepurpuric macules, developing into hemorrhagiclesions (0.5 to 2 mm) after 6 h.

b) Shw-r. The preparatory dose of 7.5 mg LPS s.c.led to infiltration of the ear. Within 2 to 3 h after


challenge with 150 mg LPS i.p. (24 h) the preparedear revealed several small purpuric macules (,0.5mm). LPS also caused systemic effects, includingdiarrhea and shivering, but no signs of cutaneousvasculitis at other, non-primed sites.

c) Lox. Injection of 0.2 ml spider venom led tosingle hemorrhage of about 0.5 to 1 mm after 6 h,sometimes accompanied by blistering of the ear’sskin.

d) ICD. Application of benzalkonium chloride ledto edema and infiltration without hemorrhage.

e) ICD with toxic necrosis by high concentration ofirritant. We used 50% of benzalkonium chloride inorder to cause a necrotizing inflammation with sec-ondary damage to blood vessels. This led to mass-

Figure 3. Histology of the Art-r, the Shw-r, Lox, and necrotizing ICD. Paraffin sections, hematoxylin–eosin. a) Art-r after 3 h:granulocytic infiltrate with leukocytoclasia, concentrated around a degenerating, thrombotic vessel; extravasation of erythrocytes.Scale bar 20 mm. b) Shw-r 3 h after challenge with LPS i.p.: fibrinoid degeneration of the vessel wall, more diffuse granulocyticinfiltrate with leukocytoclasia and extravasation of erythrocytes. Scale bar 20 mm. c) Lox after 2 h: lining of leukocytes along thevascular wall. Scale bar 20 mm. d) Necrotizing ICD 24 h after application of benzalkoniumchloride 50%: large necrobiotic area withonly few infiltrating granulocytes. At the left margin fibrinoid vessel wall with leukocytoclasia. Scale bar 20 mm.

395

ive edema and partially hemorrhagic blistering ofthe skin.

Marked extravasation of FITC-BSA only inreactions with vessel damage (Fig. 2a–d)

a) Art-r. After 6 h we observed a significant in-crease of intensity (at 517 nm) compared to theuntreated ear which indicates extravasation ofFITC-BSA and damage of blood vessels (Fig. 2a).

b) Shw-r. Six h after systemic challenge with LPSi.p. the pretreated ear showed a significant increaseof intensity in contrast to non-primed sites (Fig.2b). The intensity was not as high as in the Art-r,reflecting the smaller size of hemorrhagic lesions.The unprimed ear showed a concomitant, yet

Sunderkotter et al.

weaker, increase in fluorescence intensity, sugges-tive of increased vascular permeability due to sys-temic LPS (Fig. 2b).

c) Lox. FITC measurement revealed significant ex-travasation of FITC-BSA as a sign of vessel dam-age within 6 h (Fig. 2c).

d) ICD including necrotizing ICD. ICD revealedsome extravasation of FITC-BSA indicative of in-tense vasodilatation in the course of ear swellingdue to 2.5% benzalkonium chloride. In necrotizingICD the damage of vessels in the course of generaltissue destruction led to values similar as observedin the models of primary vasculitis (Fig. 2d).

Histology: the Shw-r and Lox show LcV similar tothe Art-r

a) Art-r. We observed the typical leukocytoclasticvasculitis characterized by a granulocytic infiltrateinitially concentrated around the blood vessels,featuring leukocytoclasia, fibrinoid degenerationof small and medium-sized blood vessels (postcap-illary venules), and extravasation of erythrocytes(Fig. 3a).

b) Shw-r. The priming phase (after LPS s.c.) wascharacterized by a diffuse perivascular infiltrateand by several intravascular leukocytes lining theinner wall of intact blood vessels. After challenge

Figure 4. Immunofluorescence staining for C3. Perivascular deposits of C3 in the Art-r (3 h) a), but not in the Shw-r (3 h after LPSi.p.) b) (scale bar 14 mm) nor in the Art-r after depletion of complement by CVF (3 h) c) (scale bar 21 mm). In mice not depletedof complement infiltrating cells synthesizing or binding complement also stain positive.

396

with LPS i.p., the infiltrate became denser andshowed leukocytoclasia, which was less frequentthan in the Art-r. Vessels revealed fibrinoid ne-crosis accompanied by extravasation of erythro-cytes and, more frequently than in the Art-r,thrombotic occlusions (Fig. 3b).

c) Lox. Within 1 to 3 h after injection of the spidervenom we observed adherence of granulocytesalong intact vessels and a growing perivascular,granulocytic infiltrate (Fig. 3c). Extravasation oferythrocytes was observed after 6 h. Leukocytocla-sia was present, but not strictly related to the ex-tent of the granulocytic infiltrate.

d) ICD. In contrast to the Art-r and Shw-r theinfiltrate was more diffuse and lacked leukocyto-clasia or vessel damage.

e) Toxic necrosis by irritant. A 50% solution of theirritant led to a dense infiltrate and degenerationof a larger area of connective tissue (necrobiosis)after 12 h (Fig. 3d). At this stage we observed fib-rinoid necrosis of blood vessels in the infiltratewith extravasation of erythrocytes. Leukocytoclas-ia was also present, however, not only around ves-sels but also around the infiltrated margins of nec-robiotic areas. Thus, damage to blood vessels sec-ondary to necrotizing inflammation in certainstages is distinguishable from primary LcV by alarge tissue necrosis not found in small vessel vas-


Figure 5. Semiquantitative score for hemorrhage after depletionof complement. a) In the Art-r delayed onset, but no completeinhibition of vasculitis after pretreatment with CVF(mean∫SD, nΩ6, asterisk denotes significantly higher degree ofhemorrhage after 3 h (P,0.05). b) In the Shw-r no attenuationof vasculitis after pretreatment with CVF (mean∫SD, nΩ6).

culitis and by the different distribution of granulo-cytes or leukocytoclasia.

Systemic application of LPS 24 h after elici-tation of the Art-r of Lox or ICD did not lead toadditional signs of LcV. This confirms that LPS-specific, local features are necessary for elicitationof vasculitis in the Shw-r (14).

Depletion of leukocytes shuts down vascularnecrosis in the Art-r, Shw-r, and in Lox, but not innecrotizing ICD or after injection ofsphingomyelinase C

We depleted mice of granulocytes by g-irradiation,to investigate which tissue alterations were me-diated primarily by granulocytes and were there-fore true vasculitis. HSA and anti-HSA, LPS orLoxosceles venom did not cause a leukocytic infil-trate in leukemic mice, neither did they lead to ves-sel damage. It was noteworthy that sphingo-myelinase C, a commercially available enzyme withsimilar action as sphingomyelinase D from theLoxosceles venom and therefore a candidate forsubstitution, induced leukocytic inflammation andhemorrhage in non-irradiated mice, but alsocaused intense vessel damage in granulocytopenic

397

mice (data not shown). This indicates that thisagent causes direct insult to vessels and thereforecannot replace Loxosceles venom in the study ofleukocyte-dependent vasculitis.

Thus, only hemorrhage in the Art-r, the Shw-rand in Lox present true vasculitides in sensu stric-tu, i.e. an inflammation whose primary event isdamage to the wall of blood vessels by leukocytes.

Activation of complement is not necessary foreither model, but can initially augment the Art-r andchallenge the Shw-r in place of LPS

On immunofluorescence the Art-r, but not theShw-r or Lox, regularly revealed deposition of C3around blood vessels (Fig. 4a, b), being detectablealready after 1–2 h (also in leukocyte-depleted, ir-radiated mice). Depletion of complement by CVF(confirmed by undetectable serum levels and by therepeated absence of C3 in anti-C3 stained tissuesections over at least 24 h) erased vascular depositsof C3 (Fig. 4c) and delayed the Art-r during thefirst 3 h, but this difference was abolished after 6h (Fig. 5a). In the Shw-r (Fig. 5b), in Lox and ICD(data not shown) depletion of complement had nosignificant effect.

Activation of complement by injecting CVF innon-depleted mice, after eliciting the Art-r, Lox orICD, did not significantly augment the signs of in-flammation (hemorrhage, ear swelling). In the Shw-r, however, CVF was able to cause local LcV whengiven in the challenge phase in place of LPS. Thiseffect was abolished in mice depleted of comple-ment.

Thus, activation of complement is not manda-tory for elicitation of the Shw-r, the Art-r or Lox,but has enhancing effects in the initial Art-r and

Figure 6. Increased number of vessels expressing E-selectin andVCAM-1 in the Shw-r after 24 h. Immunohistochemical analy-sis, 24 h after applying benzalkoniumchloride (ICD), HSA andanti-HSA (Arthus) and LPS s.c. (Shwartzman). Number ofpositive vessels in a tissue area of defined size, mean∫SD. Aster-isk denotes significantly higher numbers of positive vessels inthe Shwartzman reaction (P,0.05).

Sunderkotter et al.

Figure 7. Immunohistochemical stainingwith mAb against E-selectin. After 24 hE-selectin-positive vessels are more nu-merous in the Shw-r (a) than in ICD (b)with only 2 positive endothelial cells inthe section (arrows). Scale bar, 5 mmΩ21 mm and 18 mm.

can replace LPS effects in the challenge phase ofthe Shw-r.

Immunohistochemistry reveals sustainedexpression of adhesion molecules in the Shw-r

In all four models of inflammation, more than 85%of the early infiltrate (,12 h) consisted of PMN(Mac-1- and GR-1-positive). In the Shw-r, the in-filtrate occurring shortly before elicitation of thechallenge phase is of special interest because theremust be unique features in the tissue which exclus-ively prepare it for the elicitation of local LcV aftersystemic LPS. There were no detectable differencesin the composition of the infiltrate compared tothe other models 24 h after the initial elicitationwith regard to F4/80 (10∫4.9% F4/80-positive cellsafter LPS s.c., 15.1∫5% in the Art-r and 17.6∫5%in ICD) or Lyt-1-positive lymphocytes (data notshown). Thus, the phenotypic composition of theinfiltrate does not explain why the LPS-treatedtissue is selectively primed for LcV. However, therecould be differences in the activation status of in-filtrating leukocytes or endothelial cells.

On histology one trait of the Shw-r and Lox wasthe higher frequency of leukocytes adhering to theluminal side of endothelial cells. We therefore com-pared the sequential expression of endothelial ad-hesion molecules. In the three vasculitic reactionsthe initial expression pattern of E-selectin andVCAM-1 was similar as in ICD and as described forother non-vasculitic inflammation, i.e. a peak of ex-pression after 6 h and 10 h, resp., and a strong de-cline thereafter (11, 12, 15). However, in the Shw-r,the expression of E-selectin was more intense andmore sustained. After 24 h, prior to the challengephase, the number of positive vessels had not de-clined as in the Art-r, ICD (Fig. 6, 7a, 7b) or in Lox.Likewise expression of VCAM-1 remained mark-edly more sustained than in the other three inflam-mation models (Fig. 6). Several vessels in normalears already constitutively expressed ICAM-1, butstaining was more intense in the Shw-r.

Thus, specific priming of local tissue for theShw-r is associated with prolonged expression of

398

E-selectin and VCAM-1 rather than differences inthe composition of the infiltrate.

Flow cytometry shows increased expression ofMac-1 and ICAM-1 by circulating granulocytesin the Shw-r, but not in the Art-r or ICD

We next analysed the expression of adhesion recep-tors on leukocytes ex vivo in the different models.Mac-1 has been reported to be upregulated by LPS(16), but because of the sustained expression ofother endothelial adhesion molecules in the Shw-r, we also measured expression of LFA-1, anotherligand for ICAM-1, and of VLA-4, which bindsto VCAM-1; expression of murine ligands for E-selectin cannot be quantitated by FACS yet.

Mice were bled 15 min after eliciting LcV (after45 min mice with the Shw-r developed granulocy-topenia, likely due to increasing adherence of

Figure 8. Mean density of Mac-1 on granulocytes by FACSanalysis. Granulocytes were gated out (controlled by stainingwith Gr-1); mean density presented equivalents to the geometri-cal mean value of Mac-1-positive cells as compared with thecorresponding values of the isotype control. Increase of Mac-1after i.p. injection of LPS, CVF, or a cytokine cocktail in thechallenge phase of the Shw-r in normal mice, and only afterLPS, but not after CVF in complement-depleted mice; therewas no increase of CD18 on circulating PMN in the Art-r orin Lox. Asterisk* denotes significant rise in mean density com-pared to granulocytes ex vivo from untreated mice (nΩ10,P,0.05).


PMN to the vascular wall and accumulation invascular beds of other organs). We found no sig-nificant elevation in the percentage of Mac-1-posi-tive leukocytes, but a rapid increase in the meandensity of Mac-1 on individual granulocytes (Fig.8). A significant increase also occurred after injec-tion of CVF i.p. (Fig. 8), provided the mice werenot depleted of complement in which case onlyLPS was still able to increase the mean density ofMac-1 (Fig. 8). A similar increase was observed forICAM-1 on circulating granulocytes, but not forthe mean density of LFA-1 or VLA-4 (data notshown). No such changes were observed in circu-lating granulocytes taken from mice with Art-r orLox (Fig. 8).

Thus, development of vasculitis correlated withan increased expression of Mac-1 and ICAM-1 oncirculating granulocytes in the Shw-r, but not inthe Art-r, in Lox or ICD.

Cytokines inducible by LPS partially replace LPSin eliciting LcV in the local Shw-r

Since the effects of the preparatory and challeng-ing dose of LPS in the generalized (not localized)Shw-r could be partially induced by IFN-g or IL-12 (s.c.) and by IFN-g, IL-1 and TNF (i.p.) (17,

Tables 1 and 2. Effects of cytokines (Table 1) or of neutralization of cytokines (Table 2) on induction or inhibition of LcV and sustained expression of E-selectinin the Shw-r, and, for comparison in the other models of LcV. Cytokines able to prepare for LcV were also able to prolong expression of E-selectin (mean∫SD,significance given only for nØ5, designated by asterisk*) (Table 1), while neutralizing antibodies capable of impairing the preparatory LPS effects also impairedsustained expression (Table 2). Antibodies were given s.c. and/or i.p. as indicated

Table 1.

Number of E-selectinPriming by (all s.c.) Challenge by (all i.p.) Vasculitis positive vessels (¿)

LPS (7 mg) LPS (150 mg) Yes (5 of 6) 31.4∫9.3TNF-a (3¿104U) LPS (150 mg) No (0 of 4) Not doneIL1-b (3¿104U) LPS (150 mg) No (0 of 4) Not doneTNF-aπIL1-b (3¿104U each) LPS (150 mg) Yes (3 of 6) 21.3∫8.1IL12 (100 ng) LPS (150 mg) No (0 of 4, 2†) 3.3∫2.6IFN-g (1¿104U) LPS (150 mg) No (0 of 6) 4.6∫0.9*LPS (7 mg) CVF (1.2U) Yes (5 of 6) 28.1∫8.9LPS (7 mg) TNF-a (1¿105U)πIL1-b (1¿104U)πIFN-g (1¿104U) Yes (5 of 6) 29.0∫12.1

Table 2.

Priming by Neutralizing antibodies Challenge by Inhibition of Number of E-selectin(all s.c.) (s.c. or i.p.) (all i.p.) vasculitis positive vessels (¿)

LPS (7 mg) anti IFN-g (50 mg s.c.) LPS (150 mg) No (0 of 7) 26∫4.5LPS (7 mg) Anti IL12 (30 mg s.c., 70 mg i.p.) LPS (150 mg) No (0 of 7) 30.5∫0.5LPS (7 mg) Anti-IL12a (100 mg i.p.) LPS (150 mg) No (0 of 4) not doneLPS (7 mg) Anti-TNF-a (40 mg s.c.) LPS (150 mg) Yes (4 of 5) 19.4∫9.8LPS (7 mg) Anti-TNF-a (100 mg i.p.) LPS (150 mg) Yes (4 of 5) 17∫4.4*LPS (7 mg) Anti-IL1-b (50 mg s.c.) LPS (150 mg) Yes (2 of 5) 12∫2.1*

Arthus Reaction (anti-HSA) Anti-TNF-a (100 mg i.p.) No (0 of 7) Not doneArthus Reaction (anti-HSA) Anti-TNF-a (40 mg s.c.) No (0 of 5) Not doneCutaneous Loxoscelism Anti-TNF-a (100 mg i.p.) No (0 of 7) Not done

399

18) we investigated if these cytokines were able toalso replace LPS in inducing LcV in the localizedShw-r, and if in this case LcV would still correlatewith sustained expression of endothelial adhesionmolecules.

When we used IL-12 s.c. (10 ng, 20 ng or 100ng) or 105 U IFN-g s.c. as the preparatory dose,subsequent injection of 150 mg LPS did not resultin hemorrhage or local LcV at the site of priming(summarized in Table 1). Two of the 4 mice with100 ng IL-12 died after 12 h, confirming previousresults on systemic symptoms (17, 18). When3¿104 TNF-a and 3¿104 IL-1b were injected intothe ear, subsequent injection of 150 mg LPS i.p. ledto LcV in 50% (3 of 6) of mice. Higher concen-trations of TNF-a or IL-1b often caused localhemorrhage already before systemic injection ofLPS, as regularly observed with higher LPS doses.Immunohistochemically there was no significantlysustained expression of E-selectin (Table 1) andVCAM-1 after injection of IL-12 or IFN-g (Table1), but after injection of 3¿104 TNF-a and 3¿104

IL-1b.Thus, potency or incapability of cytokines to

prime for LcV in the Shw-r correlated with the po-tency or incapability to induce sustained expres-sion of adhesion molecules.

Sunderkotter et al.

The attempt to prevent vasculitis in the Shw-rby using neutralizing antibodies against cytokinesyielded corresponding results. Antibodies weregiven 1 h before priming with LPS. Anti-IL-12 andanti-IFN-g did not inhibit LPS-induced primingfor LcV nor did they inhibit sustained expressionof adhesion molecules (Table 2). Results with anti-IL-1b were inconsistent, but anti-TNF-a whengiven s.c. or i.p., inhibited elicitation of the Shw-rand also the sustained expression of E-selectin(Table 2) and VCAM-1 in 4 of 5 mice.

In the challenge phase of the Shw-r, only thesystemically given combination of 104 U IFN-g,

Figure 9. Semiquantitative score for hemorrhage after neutral-ization of E-selectin and VCAM-1 in the Shw-r. Injection ofeither 200 mg anti-E-selectin or anti-VCAM-1 prior eliciting thechallenge phase had no marked effects whereas simultaneousapplication of both significantly suppressed development ofhemorrhage (mean∫SD, nΩ7, asterisk * denotes significancefor P,0.05).

400

Table 3. Myeloperoxidase assay as a parameter for the amount of granulocytesand monocytes in the tissue during the Shw-r. Myeloperoxidase activity wasmeasured in ears of 6 mice in which the Shw-r was elicited shortly after theyhad been treated with anti-E-selectin, anti-VCAM-1 or both antibodies. It wassignificantly lower in ears of mice treated with anti-VCAM-1 (asterisk *), with-out or with addition of anti-E-selectin, but only in the latter case was theresuppression of hemorrhage (mean∫SD, P,0.05)

Anti E-selectinπControl Anti E-selectin Anti VCAM-1 anti VCAM-1

¿1.60 (∫0.3) ¿1.70 (∫0.5)¿2.90 (∫0.3) ¿1.65 (∫0.7)*¿2.82 (∫0.7) 1.70 (∫0.4)*

105 U IL-1b and 2 mg TNF-a (17) was able to re-place LPS i.p. (Table 1) and also increased meandensity of CD18 on circulating PMN. Here weonly neutralized with anti-TNF-a. When givenboth prior to the priming and challenging dose ofLPS, it inhibited LcV and the systemic effects ofLPS. When given only prior to the challengingdose of LPS anti-TNF-a inhibited systemic effects,but in 4 of 5 cases it did not inhibit LcV.

Thus, TNF-a is mandatory, but not sufficientfor priming and eliciting the Shw-r.

Application of anti-TNF-a before elicitation ofthe inflammatory reactions was able to reduce earswelling in ICD, in the Art-r and in Lox, howeverit did not markedly reduce hemorrhage (Table 2).

The combined neutralization of E-selectin andVCAM-1 impairs the local Shw-r

Since sustained expression of E-selectin correlatedwith elicitation of LcV in the Shw-r we attemptedto inhibit the Shw-r by injecting neutralizing anti-bodies to E-selectin. Injection of 200 mg anti-E-selectin prior to the challenge phase did not inhibithemorrhage, neither did injection of 200 mg anti-VCAM-1 when compared to mice injected withcontrol IgM or IgG (Fig. 9). However, the com-bined injection of 200 mg anti-E-selectin and anti-VCAM-1 significantly reduced local hemorrhage(Fig. 9). The administration of both antibodieswas associated with a reduction of peroxidase ac-tivity in the tissue, indicating a decrease of gran-ulocytes and monocytes (Table 3). However, as thisdecrease was less marked than the decrease ofhemorrhage and as it also occurred after injectionof anti-VCAM-1 without suppression of vasculardamage it cannot be the only cause for inhibitionof LcV.

Thus the sustained expression of E-selectin andVCAM-1 is a mandatory prerequisite for the fulldevelopment of LcV in the local Shw-r and itsmode of action in LcV is not confined to recruit-ment of leukocytes.


Discussion

Our study shows that the characteristic clinical orhistological features of leukocytoclastic vasculitis(LcV) in the skin are elicited by different agentsinducing different pathophysiological pathways.The LPS-induced local Shw-r results in LcV, whichis dependent on the combined action of cytokineswith or without complement and on the sustainedexpression of endothelial adhesion molecules inthe preparatory phase as well as increased densityof Mac-1 on circulating granulocytes in the chal-lenge phase. The immune-complex-mediated Art-rand cutaneous Loxoscelism were not dependent onrelease of TNF-a complement (though the latterincreased the Art-r initially) or on a specific ex-pression pattern of adhesion molecules. Thus, interms of these parameters they revealed no charac-teristic difference to an acute, non-vasculitic in-flammation such as ICD.

Our study also underlines that vascular injurydoes not always result from vasculitis in sensustrictu, defined as an inflammation in which de-struction of the wall of blood vessels by leukocytespresents the primary event (1). We used g-ir-radiation to deprive mice from granulocytes andwhile this procedure may not completely eliminateall granulocytes, it dramatically reduces their num-ber in the circulation so that we did not observe aninfiltrate within 24 h in any of our models. Severevascular damage in the Art-r, Shw-r and in Loxwas dependent on the presence of leukocytes as de-scribed for other species (14, 19). However, admin-istration of a highly concentrated irritant or ofsphingomyelinase C caused vascular damage alsoin neutropenic mice indicating a direct cytopathiceffect on endothelial cells. As vessel damage wasmore marked when granulocytes were present, theyappear to enhance fibrinoid necrosis of blood ves-sels in pre-damaged tissue. During toxic necrosis oftissue components the histological picture revealedleukocytoclasia and vessel damage similar to LcV.However, in LcV tissue destruction was not asmarked and diffuse as in toxic necrosis and theinfiltrate was dense in areas with tissue injury,whereas it was sparse in the necrobiotic area oftoxic necrosis. Similarly, human cutaneous LcV isusually limited to postcapillary venules of the sub-papillar plexus and, in contrast to severe toxic der-matitis, not associated with extensive tissue ne-crosis. When tissue necrosis occurs in cutaneousvasculitis of, e.g. medium sized vessels (e.g. in poly-arteriitis nodosa), it is related to the location ofthe inflamed vessel.

The differences in PMN-dependent vessel dam-age in vivo between sphingomyelinase D, one majoractive component of the Loxosceles venom, and

401

the commercially available enzyme sphingo-myelinase C, may be explained by their differentcatalytic activity. Whereas sphingomyelinase Dyields ceramide-1-phosphate without increasing in-tracellular levels of ceramide (and without directlyactivating leukocytes), treatment with sphingo-myelinase C metabolizes sphingomyelin to ceram-ide which has been shown to lead to activation ofleukocytes ((5), T. M. McIntyre, personal com-munication) and to apoptosis of bovine endo-thelial cells (20). This direct effect may be the rea-son why sphingomyelinase C causes vessel damagewithout PMN. In contrast, venom from Loxoscelesreclusa does not induce apoptosis or activatePMN, but in vitro activates endothelial cells in avery distinct way so that they cause a dysregulatedPMN response in which adhesion and degranula-tion of PMN are dissociated from shape changeand transmigration (5). Such a disturbed trans-migration of activated PMN could be one decisivecondition for PMN-mediated vasculitis in our invivo model.

A characteristic difference between our modelsof inflammation was the vascular deposition of C3,which was constant only in the Art-r. Our obser-vation that depletion of complement did not abol-ish the Art-r, is in contrast to original experimentsin rabbits and guinea pigs (19, 21), which also useddepletion via CVF. We cannot exclude that tracesof complement were still present, e.g. released bymacrophages, but since complement could not bedetected in serum or tissue of mice treated withCVF, we conclude that our treatment with CVFhad erased physiologically relevant levels of com-plement in these mice. Recent studies using C3-and FcgR-deficient mice (22) have revealed thatthe necessity of complement for the Art-r is partlydependent on the tissue site (in immune complex-induced peritonitis less than in skin and lung), onspecies and inbred strains of species (in rats morethan in mice, in 129SV mice more than in C57Bl/6 mice) (23–25). One of the crucial steps in elicitingthe Art-r rather seems to be the direct activationof leukocytes by binding of immune complexes totheir FcgRIII, FcgRI (22) without the inhibitoryeffects of FcgRII (26). Reports about the effects ofcomplement depletion on the Shw-r or on LPS-induced inflammations are still controversial.Whether it was inhibited (27, 28) or not (16, 28,29) seems to depend on the degree and method ofdepletion, on the parameters investigated (fibrino-lysis, lethality) and on the kind of Shw-r of LPS-induced inflammation. CVF was reported to re-duce hemorrhage in a model of a Shw-r in whichTNF-a was used for challenge instead of the moremultifunctional LPS (30). LPS is known to activatethe complement system (31), and activation of

Sunderkotter et al.

complement can substitute for LPS in the chal-lenge phase (our results, (32)). Yet, similarly as inthe Art-r, activation of complement was notmandatory in our study. The reason is that LPSmediates local vasculitis also by other mechanisms,such as release of TNF-a. Serum TNF-a rosemarkedly in response to LPS also in mice depletedof complement, while antiserum against TNF-awas able to inhibit the Shw-r ((13), Seeliger et al.,manuscript in preparation).

The constitutive expression of ICAM-1 andthe early temporal sequence in the expression ofE-selectin and VCAM-1 were similar in the Art-r, the localized Shw-r and in ICD. This finding isconsistent with observations made in othermouse models of inflammation with regard toICAM-1 (33) E-selectin (12, 15) and VCAM-1(11, 15). Thus, the initial expression pattern ofadhesion molecules appears to be a process com-mon to all acute inflammatory tissue reactionsand does not present a characteristic feature forvasculitis. However, the sustained expression ofVCAM-1 and especially of E-selectin in the Shw-r has not been reported for other inflammations.E-selectin is a known, albeit not the only ligandfor granulocytes (10, 34). VCAM-1 also has beenshown to mediate diapedesis of granulocytes (35,36). The presence or absence of E-selectin andVCAM-1-positive vessels 24 h into the prepara-tory phase correlated tightly with the presence orabsence of LcV in the Shw-r in those experi-ments where cytokines were neutralized or ad-ministered in place of LPS. We confirmed thatthe sustained expression of both adhesion mol-ecules is a prerequisite for induction of LcV inthe Shw-r as we could markedly suppress hemor-rhage when we injected neutralizing antibodiesagainst both adhesion molecules. Curiously, nei-ther antibody had suppressive effects when usedalone and anti-E-selectin tended to increasehemorrhage in 2 of 6 mice. In another study anantibody against E-selectin markedly exacerbatedendothelial injury in lungs in the generalized mu-rine Shw-r in mice (37). However, the pathophys-iology between cutaneous LcV in the local Shw-rand systemic hemorrhage in the generalized Shw-r may differ and the antibody used by theseauthors has only weak neutralizing effects in vivo((10), our own unpublished data) in contrast tothe one used in our experiments (12). The inhi-bition of the local Shw-r by the simultaneous in-jection of both antibodies did not only resultfrom a reduction of leukocytes, as a similar re-duction after injection of anti-VCAM-1 was notsufficient to suppress vascular damage. However,as these adhesion molecules may not only me-diate rolling or adherence of PMN in LcV, but

402

also signaling for further steps in diapedesis orfor release of toxic cell products (34–36, 38) theirsustained expression may interfere with the se-quence of events necessary for undisturbed diap-edesis of leukocytes in non-vasculitic inflam-mations (35–40). LcV in the Shw-r also corre-lated with upregulation of counter receptors forICAM-1 on granulocytes after challenge withLPS and CVF, similarly as observed in rats (16).It is likely that injection of LPS in the challengephase resulted in activation and increased ad-hesiveness of CR3, either by direct effect of LPS(41) or by e.g. IL-8 from activated endothelialcells (34). LPS and products of complement acti-vation have been known to increase the cytotoxicpotential of leukocytes (13, 32, 42). Degranula-tion of PMN was increased when PMN adheredto a surface via CD18 (43). As Mac-1 reachesthe cell surface in the course of degranulation,our results indicate that there was marked de-granulation of circulating PMN shortly after sys-temic administration of LPS. Thus, one conceptfor the pathophysiology of LcV in the Shw-rwould encompass that the sustained expressionof E-selectin and VCAM-1 at the site of primingresults in raised number of PMN adherent to thevascular wall, which are then activated by sys-temic LPS to adhere more tightly and, at thesame time, to degranulate close to endothelialcells, releasing cytotoxic substances. Neutraliza-tion of Mac-1 or genetic deletion of ICAM-1 hasalso been found to inhibit LPS-induced inflam-mations in different species (32, 44–46).

When comparing the three models of leukocy-toclastic vasculitis with non-vasculitic ICD, weconclude that LcV could result from a constel-lation in which activation of PMN (e.g. by engage-ment of FcR or by LPS and complement productsor products of endothelium distinctly activated byLoxosceles venom) is linked to vascular events in-terfering with normal diapedesis (e.g. deposition offixed immune complexes, sustained expression ofadhesion molecules, dysregulated activation byLoxosceles venom). Such a constellation differsfrom non-vasculitic inflammations and may directthe damaging potential of granulocytes towardsthe postcapillary venule. For assessment of pa-tients with vasculitis it is important to know thatthe histological features of leukocytoclastic vas-culitis can be found in different inflammatory reac-tions which are not all caused by immune complex-es (39).

Acknowledgements

We thank Dr Rübe (Department for Radiotherapy, Universityof Münster) for irradiation of mice, Ms A. Erpenbeck, Ms A.


Wissel, Ms U. Keller, Ms C. Post, Ms M. Wendland, Ms E.Nattkemper and Ms S. Merfeld for excellent technical assist-ance. This work was supported by a grant No. So 87/11 of theDeutsche Forschungsgemeinschaft and by the Clinical ResearchCenter, Münster (D15).

References

1. Sunderkötter C, Kolde G. Cutaneous vasculitis. In: Bos J,ed. The Skin Immune System (SIS) Basic and Clinical Im-munodermatology. Ed 2, Boca Raton, Florida, USA: CRCPress, 1997: 479–488.

2. Jennette C J, Milling D M, Falk R J. Vasculitis affectingthe skin. Arch Dermatol 1994: 130: 899–906.

3. Lentsch A B, Ward P A. Regulation of inflammatory vascu-lar damage. J Pathol 2000: 190: 343–348.

4. Sams H H, Dunnick C A, Smith W L, King L E. Necroticarachnidism. J Am Acad Dermatol 2001: 44: 561–573.

5. Patel K D, Modur V, Zimmerman G A, Prescott S M,McIntyre T M. The necrotic venom of the brown reclusespider induces dysregulated endothelial cell-dependent neu-trophil activation. Differential induction of GM-CSF, IL-8, and E-selectin expression. J Clin Invest 1994: 94: 631–642.

6. Patterson C E, Rhoades R A, Garcia J G. Evans blue dyeas marker of albumin clearance in cultured endothelialmonolayer and isolated lung. J Appl Physiol 1992: 72: 865–873.

7. Baumann U, Köhl J, Tschernig T et al. A codominant roleof FcgRI/II and C5aR in the reverse Arthus reaction. JImmunol 2000: 164: 1065–1070.

8. Clauss M, Sunderkötter C, Sveinbjörnsson B et al. A per-missive role for TNF in VEGF-induced vascular per-meability. Blood 2001: 97: 1321–1329.

9. Henseleit U, Steinbrink K, Goebeler M et al. E-selectin ex-pression in experimental models of inflammation in mice. JPathol 1996: 180: 317–325.

10. Bosse R, Vestweber D. Only simultaneous blocking of theL- and P-selectin completely inhibits neutrophil migrationinto mouse peritoneum. Eur J Immunol 1994: 24: 3019–3024.

11. Henseleit U, Steinbrink K, Sunderkötter C, Goebeler M,Roth J, Sorg C. Expression of murine VCAM-1 in vitro andin different models of inflammation in vivo: correlation withimmigration of monocytes. Exp Dermatol 1994: 3: 249–256. Erratum: Exp Dermatol 1995: 4: 327.

12. Austrup F, Vestweber D, Borges E et al. P- and E-selectinmediate recruitment of T-helper-1 but not T-helper-2 cellsinto inflamed tissues. Nature 1997: 385: 81–83.

13. Warren J S, Yabroff K R, Remick D G et al. Tumor necrosisfactor participates in the pathogenesis of acute immunecomplex alveolitis in the rat. J Clin Invest 1989: 84: 1873–1882.

14. Brozna J P. Shwartzman reaction. In: Seminars in Throm-bosis and Homeostasis. New York: Thieme Medical Pub-lishers Inc, vol. 16, 1990: 325–332.

15. Fries J W U, Williams A J, Atkins R C, Newman W, Lip-scomb B F, Collins T. Expression of VCAM-1 and E-se-lectin in an in vivo model of endothelial activation. Am JPathol 1993: 143: 725–737.

16. Witthaut R, Farhood A, Smith C W, Jaeschke H. Comple-ment and tumor necrosis factor-a contribute to Mac-1(CD11b/CD18) up-regulation and systemic neutrophil acti-vation during endotoxemia in vivo. J Leukocyte Biol 1994:55: 105–111.

17. Ozmen L, Pericin M, Hakimi J et al. Interleukin 12, Inter-feron g, and tumor necrosis factor a are the key cytokinesof the generalized Shwartzman reaction. J Exp Med 1994:180: 907–915.

403

18. Ogasawara K, Takeda K, Hashimoto W et al. Involvementof NK1π T cells and their IFN-g production in the general-ized Shwartzman reaction. J Immunol 1998: 160: 3522–3527.

19. Cochrane C G, Müller-Eberhard H J, Aikin B S. Depletionof plasma complement in vivo by a protein of cobra venom:its effect on various immunologic reactions. J Immunol1970: 105: 55–69.

20. Haimowitz-Friedman A, Cordon-Cardo C, Bayoumy S etal. Lipopolysaccharide induces disseminated endothelialapoptosis requiring ceramide generation. J Exp Med 1997:186: 1831–1841.

21. Ward P A, Cochrane C G. Bound complement and immun-ologic injury of blood vessels. J Exp Med 1965: 212: 215–237.

22. Ravetch J V, Clynes R A. Divergent roles for Fc receptorsand complement in vivo. Annu Rev Immunol 1998: 16: 421–432.

23. Szalai A J, Digerness S B, Agrawal A et al. The Arthusreaction in rodents: Species-specific requirement of comple-ment. J Immunol 2000: 164: 463–468.

24. Hazenbos W L, Gessner J E, Hofhuis F M et al. ImpairedIgG-dependent anaphylaxis and Arthus reaction in Fcgamma RIII (CD16) deficient mice. Immunity 1996: 5: 181–188.

25. Köhl J, Gessner E. On the role of complement and Fc g-receptors in the Arthus reaction. Mol Immunol 1999: 36:893–903.

26. Ierino F L, Powell M S, McKenzie I F C, Hogarth P M.Recombinant soluble human FcgRII: Production, char-acterization, and inhibition of the Arthus reaction. J ExpMed 1993: 178: 1617–1628.

27. Fong J S C, Good R A. Prevention of the localized andgeneralized Shwartzman reactions by an anticomplementa-ry agent, cobra venom factor. J Exp Med 1971: 134: 642–655.

28. Polak L, Turk J L. Suppression of the haemorrhagic com-ponent of the Shwartzman reaction by anti-complementserum. Nature 1969: 223: 738–739.

29. Bergstein J M, Michael A F. Failure of cobra venom factorto prevent the generalized Shwartzman reaction and loss ofrenal cortical fibrinolytic activity. Am J Pathol 1974: 74:19–30.

30. Attiyah R A, Rosen H, Rook G A W. A model for theinvestigation of factors influencing haemorrhagic necrosismediated by tumour necrosis factor in tissue sites primedwith mycobacterial antigen preparations. Clin Exp Immun-ol 1992: 88: 537–542.

31. Philips M, Abramson S, Weissmann G. Neutrophil ad-hesion and autoimmune vascular injury. Clin Aspects Auto-immun 1989: 3: 6–15.

32. Argenbright L W, Barton R W. Interactions of leukocyteintegrins with intercellular adhesion molecule 1 in the pro-duction of inflammatory vascular injury in vivo. J Clin In-vest 1992: 89: 259–272.

33. Goebeler M, Gutwald J, Roth J, Meinardus-Hager G, SorgC. Expression of intercellular adhesion molecule-1 in mu-rine allergic contact dermatitis. Int Arch Allergy Appl Im-munol 1990: 93: 294–299.

34. Springer T A. Traffic signals for lymphocyte recirculationand leukocyte emigration: the multistep paradigm. Cell1994: 76: 301–314.

35. Taooka Y, Chen J, Yednock T, Sheppard D. The integrinalpha9beta1 mediates adhesion to activated endothelialcells and transendothelial neutrophil migration through in-teraction with vascular cell adhesion molecule-1. J Cell Biol1999: 145: 413–420.

36. Davenpeck K L, Sterbinsky S A, Bochner B S. Rat neutro-phils express alpha4 and beta1 integrins and bind to vascu-

Sunderkotter et al.

lar cell adhesion molecule-1 (VCAM-1) and mucosal ad-dressin cell adhesion molecule-1 (MAdCAM-1). Blood1998: 91: 2341–2346.

37. Koide N, Kato Y, Sugiyama T et al. Exacerbation of vascu-lar endothelial injury in the generalized Shwartzman reac-tion by the administration of anti-E-selectin antibody.Microbiol-Immunol 2000: 44: 197–200.

38. Nagata M, Sedgwick J B, Vrtis R, Busse W W. Endothelialcells upregulate eosinophil superoxide generation viaVCAM-1 expression. Clin Exp Allergy 1999: 29: 550–561.

39. Belmont H M, Abramson S B, Lie J T. Pathology andpathogenesis of vascular injury in systemic lupus ery-thematosus. Arthritis Rheum 1996: 39: 9–22.

40. Vestweber D. Molecular mechanisms that control endo-thelial cell contacts. J Pathol 2000: 190: 281–291.

41. Wright S D, Ramos R A, Hermanowski-Vosatka A,Rockwell P, Detmers P A. Activation of the adhesive ca-pacity of CR3 on neutrophils by endotoxin: dependence onlipopolysaccharide binding protein and CD14. J Exp Med1991: 173: 1281–1286.

404

42. Zhang X, Morrison D C. Lipopolysaccharide-inducedselective priming effects on tumor necrosis factor a andnitric oxide production in mouse peritoneal macrophages.J Exp Med 1993: 177: 511–516.

43. Nathan C F. Neutrophil activation on biological surfaces.J Clin Invest 1987: 80: 1550–1560.

44. Lum H, Gibbs L, Lai L, Malik A B. CD18 integrin-de-pendent endothelial injury: effects of opsonized zymosanand phorbol ester activation. J Leukocyte Biol 1994: 55:58–63.

45. Toh H, Miyata T, Torisu M. Characterization of neutrophilactivation by repeated injection of endotoxin in rabbits.Role of neutrophils in the generalized Shwartzman reac-tion. J Leukocyte Biol 1993: 53: 256–263.

46. Sligh J E Jr, Ballantyne C M, Rich S S et al. Inflammatoryand immune responses are impaired in mice deficient in in-tercellular adhesion molecule 1. Proc Natl Acad Sci USA1993: 90: 8529–8533.

different pathways leading to cutaneous leukocytoclastic vasculitis in mice

Documents