Annals of the Rheumatic Diseases 1991; 50: 946-949

Deposition of eosinophil cationic protein in vascularlesions in temporal arteritis

Roger Hallgren, Bjorn Gudbjornsson, Erik Larsson, Kjeld Fredens

AbstractThe possible role of the eosinophil and itscytotoxic granule proteins in the vascularlesions seen in temporal arteritis was eluci-dated. Sixteen sections of biopsy specimensfrom arteria temporalis showing giant cellarteritis were stained for eosinophil cationicprotein (ECP) by polyclonal antibodies andthe immunoperoxidase method. Activatedeosinophils were identified by monoclonalantibodies linked to alkaline phosphatase.Activated eosinophils and secreted ECP wereseen in all layers of the inflamed vessels andwere most evident in necrotic lesions andthrombi. Only a small number of granulocytesseen in the adventitia were immunoreactivefor cathepsin G, and no extracellular depositsof this neutrophil granule protein were seen.A few immunoreactive eosinophils were foundin the adventitia in two of five negativetemporal artery biopsy specimens frompatients with polymyalgia rheumatica. Alleight coronary artery biopsy specimens withatherosclerotic lesions showed no activatedeosinophils or secreted ECP. These findingsindicate that eosinophils are involved in thevascular lesion in temporal arteritis and sug-gest that cytotoxic eosinophil granule proteinsmay contribute to the necrotic lesions andthe development of thrombi.

Departments of InternalMedicine and Pathology,University Hospital,Uppsala, Sweden andDepartment of Anatomy,University of Aarhus,Aarhus, DenmarkR HallgrenB GudbjornssonE LarssonK FredensCorrespondence to:Dr Roger Haligren,Section of Rheumatology,Department of InternalMedicine, UniversityHospital, S-751 85 Uppsala,Sweden.

Accepted for publication30 November 1990

Giant cell arteritis in temporal arteritis-polymyalgia rheumatica is characterised bypatchy necrotic lesions and perivascular infil-trates of, predominantly, histiocytes, lympho-cytes, epithelioid cells, and giant cells, whichgive the lesion its traditional name. The lumenis markedly narrowed by the thickened oede-matous intima and at times occluded bythrombi. The possible role of the eosinophil inthe vascular lesion in temporal arteritis has notbeen considered before, as far as we know,partly owing to the limited attention the eosino-phil has received in general in inflammatorylesions. Recent findings, however, have sug-gested that eosinophils may be involved in thevascular injury associated with the hyper-eosinophilic syndrome' and the eosinophilicgranulomatous angiitis of Churg and Strauss.2The cytotoxic properties of the major eosinophilgranule proteins, eosinophil cationic protein(ECP)3 and major basic protein,' 7 8 suggestedthat their extracellular deposition may contri-bute to vascular injury. The activity of ECP onfactor XII dependent coagulation pathways9and its neutralising effect on the anticoagulanteffect of heparin3 may favour the development

of thrombi in necrotic vascular lesions. In thisstudy we report that temporal arteritis shouldbe added to the list of vascular diseases charac-terised by an infiltration of activated eosino-phils and extracellular deposits of ECP withinthe necrotic and thrombotic lesions.

Patients and methodsBiopsy specimens of the temporal artery show-ing histological signs of giant cell arteritis wereobtained from 16 patients (three male, 13female; mean age 69 years, range 56-79) beforethe institution of corticosteroids. Negativetemporal artery biopsy specimens from fivepatients with polymyalgia rheumatica andcoronary artery biopsy specimens from eightpatients undergoing coronary bypass surgerywere obtained for control studies. The biopsyspecimens were fixed in a 4% solution withbuffered formaldehyde and embedded inparaffin. Serial sections were studied; the firstsections were stained according to routine pro-cedures (haematoxylin/eosin) and the followingsections were used for immunohistochemistry.Paraffin sections were dewaxed, rehydrated,and subjected to immunostaining with theantibodies used.The antibodies used for immunohisto-

chemistry were polyclonal antibodies againstECP'0 and a monoclonal antibody, EG2, directedagainst an epitope on the secreted form ofECP. " This antibody recognises activatedeosinophils." 12 To compare eosinophil re-activity with that of the neutrophil an additionalantibody against cathepsin G (chymotrypsin-like cationic protein) was included.'0 Theimmunoperoxidase method according toSternberger'3 was used with polyclonal anti-bodies against ECP and cathepsin G. Thestaining procedures with these polyclonal anti-bodies have previously been described indetail. 14 The monoclonal antibody EG2 wasdetected by the unlabelled bridge technique(APAAP procedure). 14 A mouse monoclonalantibody of the same isotope was used ascontrol. Conventional staining controls wereused'3 and specificity controls performed byblocking the reactivity of the antisera withpurified ECP. The immune reactivities of cell-bound and extracellular ECP were dis-tinguished. '4 '5

ResultsImmunohistochemical staining of the 16 biopsyspecimens displaying temporal arteritis showedan infiltration of granulocytes and extracellular

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Eosinophil activation in temporal arteritis

deposits reactive towards the polyclonal ECPantibody and the monoclonal antibody EG2(fig 1). Most, if not all, cells and depositsreactive towards the polyclonal ECP antibodywere also strongly EG2 positive. In contrast,only a few cells positive for cathepsin G wereseen in the adventitia and in eight specimens nocells immunoreactive for cathepsin G werepresent at all. The number of eosinophils in thecell infiltrates seen by haematoxylin-eosin stain

i , ,- ,r

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Figure I Section oftemporal artery with giant cell arteritis. Cells immunoreactiveeosinophil cationic protein (ECP) infiltrate the media. Deposits ofextracellularECin these areas and are indicated by the small arrows. L indicates the lumen and the hthe membrana elastica interna. In the adventitia (adv) only afew cells immunoreacECP are seen (counterstained with toluidine).

i. ~ ~

''' +

Figure 2 Section oftemporal arteryfrom another patient with typical temporal art4Cells immunoreactive for eosinophil cationic protein (ECP) infiltrate the intima, an,

granular deposits ofECP are seen (small arrows). Extensve ECP deposits are seen

thrombus (asterisk). The large arrow indicates the mebrana elastica innterna. Coun,with toluidine.

was considerably lower than the number identi-fied by immunofluorescence.

Immunoreactivity was rated according to thescale: O=none, 1 =dim, 2=moderately bright,3= bright. In all biopsy specimens reactivity forECP was located to cells infiltrating the adven-titia. Six biopsy specimens had a score of 1, six ascore of 2, and four a score of 3. In 12/16 biopsyspecimens cells reactive with ECP were seen inthe media; nine of these specimens had a scoreof 1 and three a score of 3. The immunoreactivecells were localised in areas with round cellinfiltration. In 10/16 biopsy specimens cellsimmunoreactive for ECP were also seen in theintima (fig 2). Eight of these specimens had animmunoreactivity score of 1 and only two had ascore of 3. In all biopsy specimens patchy andmostly dim extracellular deposits of ECP wereseen in areas with immunoreactive cells. Strik-ing deposits of ECP were seen in areas withnecrotic lesions and in thrombi (fig 2).The eight coronary arteries with athero-

sclerotic lesions showed no immunoreactivityfor EG2 and no extracellular deposits of ECP.In two of five temporal artery biopsy specimens,which were otherwise negative, a small numberof cells immunoreactive for ECP were seen inthe adventitia; in one of the specimens the cellswere positive for EG2 but had no extracellulardeposits of ECP.

DiscussionThis study showed that the vascular damage intemporal arteritis is characterised by an invasionof activated eosinophils and extracellular

,Pare seen deposits of the cytotoxic eosinophil granule!arge arrow protein ECP in all layers of the inflamed vessel.tivefor Areas with necrotic lesions showed the most

extensive deposits of ECP. The discovery ofheavy ECP deposits in thrombi in necroticvascular lesions is of potential interest owing tothe effects ofECP on heparin and the coagulationpathways.4 9 In contrast, eosinophils or eosino-phil granule deposits were absent from athero-sclerotic arteries. Activated eosinophils,however, were identified in the adventitia ofotherwise negative temporal artery biopsy speci-mens from some of the patients with poly-myalgia rheumatica.These findings in temporal arteritis extend

the list of vasculitis diseases in which eosinophilsplay a part. Deposits of eosinophil granuleproducts have previously been reported in theeosinophil granulomas in Churg-Strauss syn-drome.' In renal allograft rejections of thevascular type, extensive deposits of secretedECP can be seen in those arterial walls withnecrotic lesions but not in inflamed vesselswithout necrosis. 16 Immunohistochemicalstudies of cardiac tissue of patients with eosino-philic endomyocardial disease have shown thatactivated eosinophils and secreted eosinophilgranule proteins are most evident within thenecrotic and, at a later stage, thrombotic lesionsof the myocardium, within areas of acute tissue

nertis. damage in the endocardium and in the walls ofd heavy, small vessels.' '4 Thus eosinophil activation istetina d not specific for certain vasculitic diseases which

by tradition have been associated with eosinophil

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Hallgren, Gudbjornsson, Larsson, Fredens

involvement but seems to be present in a varietyof inflammatory diseases affecting vessel walls.Most theories of eosinophil function have

been related to allergic diseases and parasiteinfections. A modulatory role for eosinophils inallergic reactions has been proposed, and somesupport for the hypothesis has been provided.Thus eosinophils may exert a modulating effecton the inflammatory reaction by release of arylsulphatase B, phospholipase D, histaminase,and other chemical mediators. 17-19 The idea ofthe eosinophil as an innocent bystander ormodulator of the inflammatory reaction hasbeen questioned during recent years, however,mainly owing to the identification of the cyto-toxic properties of the eosinophil granule con-stituents ECP, major basic protein, and eosino-phil peroxidase.8 The ability of eosinophils tokill parasites has been attributed to thesegranular cytotoxins.5-7 As both ECP and majorbasic protein are highly toxic to several mam-malian cells and tissues in vitro5 8 it has beenproposed that these proteins when secreted maycause tissue damage. The hypothesis that theeosinophil may be turned against the host hasbeen supported not only by the findings innecrotising vasculitides2 or myocardial lesionsin hypereosinophilic syndrome' 14 but also byobservations made on eosinophil involvementin, for example, mucosal damage in coeliacdisease,'5 skin damage in atopic eczema,20impaired lung function in certain interstitiallung diseases,2' 22 and the inflammatory jointlesion.The cause of temporal arteritis is unknown.

The predilection of temporal arteritis for thewhite population is striking. This predilectionand the appearance of the disease in members ofthe same family suggested a genetic pre-disposition.24 The pathogenic mechanismsimplicated in the vascular accumulation ofround cells and giant cells are also unknown.Characterisation of the infiltrating lymphocyteshas shown that most are activated T lympho-cytes.2' The mechanisms behind the localrecruitment and activation of eosinophils intemporal arteritis have not been identified inthis study but certain links exist between theactivation of lymphocytes and eosinophils.Primed clones of lymphocytes in the presence ofspecific antigens induce a local accumulationof eosinophils,26 possibly by generation of aneosinophilic chemotatic factor.2729 The needfor a T cell function in the eosinophil responseto certain antigens has been clearly shown.30 Anumber of endogenous factors released fromstimulated lymphocytes and monocytes arereported to augment several of the effectorproperties ofhuman eosinophils.3' In particular,interleukin 5 seems to have a large potential toamplify the proliferative response of eosinophilsand to induce eosinophil activation.32 Theselymphocyte dependent mechanisms may beoperative not only in temporal arteritis but alsoin most of the other clinical situations with signsof local eosinophil accumulation and activation.

Neutrophil granulocytes may also participatein inflammatory tissue injury by their ability torelease enzymes and toxic oxygen radicals.33 34The azurophilic granules of the neutrophil

contain a number of preformed proteolyticenzymes, including cathepsin G, which have theability to damage the connective tissue matrix.In the vascular lesion in temporal arteritis theabsence of neutrophil infiltration was striking-only a few granulocytes showed immuno-reactivity for cathepsin G and no extracellulardeposits of this granule protein were seen. Thusconventional staining seems to underestimatethe presence of eosinophils in the leucocyteinfiltrate owing to the fact that degranulatingeosinophils do not stain well with eosin having ahigh affinity for the eosinophilic granules.The possible mechanism by which ECP

induces cell damage has been elucidated byYoung et al.35 They showed that ECP inducesnon-ion selective pores in the cellular mem-branes and proposed that cell damage wasinduced by a colloid-osmotic process. Thusdeposits of extracellular ECP in the vessel wallsin temporal arteritis may, by such a mechanism,contribute to the vascular damage induced byinflammation. However, even though ECP is apotent cytotoxin, the immunohistochemicalvisualisation of ECP in injured tissue gives noquantitative estimate of the deposited amounts.Furthermore, interpretation of the possiblepathophysiological role of secreted eosinophilgranule proteins is hampered by a lack of infor-mation about the turnover ofECP in tissue. Thetheoretical possibility that the cytotoxic propertyof ECP in tissue may become neutralised has tobe considered as well. Thus although some ofthe clinical observations made, including thepresent one in temporal arteritis, are intriguing,suggesting that eosinophils play a part in tissuedamage, the possibility that eosinophils mayhave a modulatory role on inflammation cannotbe excluded.

We thank Dr P Venge, University Hospital, Uppsala, Sweden forsupplying the purified eosinophil cationic protein. This studywas supported by the Swedish Medical Research Council,Svenska Livforsikringsbolags namnd for medicinsk forskning,Riksforeningen mot reumatism and Nationalforeningen,Copenhagen.

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