J Clin Pathol 1994;47:939-944

Comparison of cell adhesion molecule expressionin cutaneous leucocytoclastic and lymphocyticvasculitis

N P Burrows, F A Molina, G Terenghi, P K Clark, D 0 Haskard, JM Polak, R R Jones

AbstractAims-To compare the expression of thecell adhesion molecules intercellularadhesion molecule-I (ICAM-1), ELAM-1(E-selectin), and vascular cell adhesionmolecule-i (VCAM-1) in cutaneous leu-cocytoclastic and lymphocytic vasculitis.Methods -Immunohistochemical analy-sis was performed on early lesional skinbiopsy specimens of leucocytoclastic vas-culitis (n = 14), lymphocytic vasculitis(n = 10), non-lesional skin (n = 12), andnormal skin (n = 5). A standard immuno-peroxidase technique was used to detectexpression of ICAM-1, E-selectin,VCAM-1, and the cell markers CD11a,CD11b, CD11c, von Willebrand factor,CD3, CD68, and neutrophil elastase(NP57).Results-Basal keratinocyte intercellularadhesion molecule-i was expressed ineight (80%) cases of lymphocytic and inonly one (7%) case of leucocytoclasticvasculitis, and not in non-lesional skin orcontrol biopsy specimens from normalsubjects. E-selectin was expressed onvascular endothelium in eight (57%)cases of leucocytoclastic and in seven(70%) cases of lymphocytic vasculitis.Endothelial vascular cell adhesion mole-cule-i expression was seen in three (21%)biopsy specimens of leucocytoclastic andfive (50%) oflymphocytic vasculitis.There were increased numbers of cells

in the dermal infiltrate stained for NP57,CD11b, and CD11c in leucocytoclasticcompared with lymphocytic vasculitis (p< 0 001, p = 0-013, p = 0 009, respec-tively); immunoreactive positive cells forCD3 and CD11a were increased in lym-phocytic compared with leucocytoclasticvasculitis (p < 0 001, p = 0 011, respec-tively).Conclusions-These observations indi-cate that upregulation of adhesion mole-cule expression occurs in bothleucocytoclastic and lymphocytic vasculi-tis. The different patterns of adhesionmolecule expression in the two groups ofvasculitis may reflect differences in thelocal release of cytokines. In particular,detection of intercellular adhesion mole-cule-i expression by keratinocytes inlymphocytic vasculitis is consistent withan active role for mediators derived fromT lymphocytes in the pathogenesis of thelesion.

( Clin Pathol 1994;47:939-944)

Cutaneous vasculitis embraces a group ofconditions with characteristic histological fea-tures involving small and medium sized bloodvessels. Leucocytoclastic vasculitis, whichmay be associated with several clinical disor-ders, is characterised by a predominance ofperivascular polymorphonuclear cells, nucleardust, red cell extravasation, and deposition offibrinoid material around cutaneous bloodvessels. In contrast, lymphocytic vasculitiscomprises a heterogeneous group of disorderscharacterised clinically by purpura and histo-logically by red cell extravasation in associa-tion with a perivascular mononuclear cellinfiltrate. Controversy still exists about thepathogenesis of cutaneous vasculitis and itis not readily apparent why some patientsexhibit a neutrophilic and others a lymphocyticinfiltrate.

Several early studies of leucocytoclastic vas-culitis have clearly shown the deposition ofimmunoglobulin and complement within ves-sel walls.' 2 In contrast, lesional deposits ofimmunoglobulin and complement are rarelyfound in patients with lymphocytic vasculitis'and cell mediated immunological mechanismshave been implicated.4-8 Massa and Su9 feltthat lymphocytic vasculitis is not a specificclinicopathological entity but a combinationof histological features seen in a variety ofclinical settings which include capillaritis,pityriasis lichenoides, toxic erythema, anddrug induced purpura. It has also beensuggested that the finding of perivascularlymphocytes in some cases of vasculitis is dueto the biopsy specimen being taken at a rela-tively late stage in the development of alesion.2 10

It is well recognised that cell adhesion mol-ecules are important in the regulation of nor-mal inflammatory responses." ICAM-1(intercellular adhesion molecule-i) belongs tothe immunoglobulin supergene family and isexpressed on several different types of cells,including endothelial cells, activated lympho-cytes, macrophages and activated ker-atinocytes."2 It is constitutively expressed onendothelial cells but it may be upregulated byinterleukin-1, tumour necrosis factor, orinterferon y."1 14 ICAM-1 binds the leucocyte112 integrins lymphocyte function associatedantigen-1 (CD11a/CD 18) and CDi lb/CD 18 which mediate leucocyte-endothelialcell adhesion and the transendothelial

Department ofHistochemistryN P BurrowsF A MolinaG TerenghiJ M PolakDepartment ofMedical PhysicsP K ClarkDepartment ofMedicineD 0 HaskardDepartment ofDermatology, RoyalPostgraduate MedicalSchool,HammersmithHospital, Du CaneRoad, LondonW12 ONNR R JonesCorrespondence to:Dr N P Burrows,Department ofDermatology,Addenbrooke's Hospital,Hills Road, Cambridge,CB2 2QQAccepted for publication9 March 1994

939

on July 24, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.10.939 on 1 O

ctober 1994. Dow

nloaded from

Burrows, Molina, Terenghi, Clark, Haskard, Polak, J7ones

migration of leucocytes into the tissues." 15VCAM-1 (vascular cell adhesion molecule-i)also belongs to the immunoglobulin super-gene family. Expression of VCAM-1 on largevessel endothelial cells is induced by inter-leukin- 1, tumour necrosis factor, or inter-leukin-4,'6-'8 whereas induction on

microvascular endothelial cells is restricted totumour necrosis factor in vitro.'9 Expressionof VCAM-1 in the skin is not restricted toendothelial cells but is also seen on some

perivascular and dermal dendritic cells.20VCAM-1 binds to VLA-4 (CD49d/CD29)found on lymphocytes, monocytes,eosinophils, and Langerhans' cells.2' E-selectin (endothelial leucocyte adhesion mole-cule-1) was first identified as an adhesionmolecule for neutrophils.22 It is now recog-nised as also binding to eosinophils,basophils, monocytes, and a subpopulation ofmemory T cells (CD4+, CD45RO).2324 Theneutrophil ligand for E-selectin has been iden-tified as a carbohydrate, sialyl-Lewis X,25whereas the cutaneous lymphocyte associatedantigen (CLA), recognised by the monoclonalantibody HECA-452, is the ligand present onmemory T cells.26 E-selectin is minimallyexpressed on unstimulated endothelial cellsbut is induced by interleukin-1 or tumournecrosis factor, but not directly by interferony or interleukin-4.'8 27

There is evidence that the patterns of celladhesion molecule expression may reflect thenature of the leucocyte infiltrate present indifferent forms of cutaneous inflammation.20To throw further light on the pathogenesis ofvasculitis, we therefore investigated whetherleucocytoclastic and lymphocytic vasculitisare associated with differential expression ofthese cell adhesion molecules.

MethodsHospital ethical committee approval was

obtained to perform lesional and, where possi-ble, site matched non-lesional elliptical biop-sies in patients with cutaneous vasculitis. Sitematched skin biopsy specimens were alsotaken from five age and sex matched healthyvolunteers. The biopsies were performedunder 2% lignocaine anaesthetic injected intothe periphery of the area to be excised.The skin specimens were bisected and one

half was fixed in formalin and paraffin wax

embedded for histological examination. Theother half was immediately fixed for immuno-histochemistry by immersion in Zamboni'sfluid for six hours at room temperature, thentransferred into a O 1M phosphate bufferedsaline (PBS, pH = 7.2) containing 15%sucrose and 0-1% sodium azide. Frozen sec-

tions (10 pum) were cut, collected onto poly-L-lysine coated glass slides, and left to dry atroom temperature for one hour before beingimmunostained.Twenty four patients with active cutaneous

vasculitis were recruited from the dermatol-ogy outpatient department. The patientswith vasculitis were subdivided, according toestablished histological criteria, into leucocy-

Table 1 Clinimcopathological diagnosesLeucocytoclastic Lymphocyticvasculitis (n = 14) vasculitis (n = 10)

Urticarial vasculitis (n = 7) Capillaritis (n = 6)Cryoglobulinaemia (n = 2) Pityriasis lichenoidesHenoch Schonlein purpura (n = 2) chronica (n = 1)Drugs (n = 1) Idiopathic (n = 3)Idiopathic (n = 2)

toclastic or lymphocytic vasculitis (table 1).28The antibodies used for staining are listed

in table 2. Sections were stained using thethree-step peroxidase-antiperoxidase (PAP)immunohistochemical method.29 Endogenousperoxidases were inhibited by soaking the sec-

tions in 0O3% hydrogen peroxide in methanolfor 15 minutes. Diaminobenzidine was usedas a substrate to visualise von Willebrand fac-tor, ICAM-1, E-selectin, and VCAM-1; thesections were counterstained with Mayer'shaematoxylin. To visualise leucocyte markersand integrin expression, the peroxidase was

developed according to the glucoseoxidase/nickel enhancement reaction.30Negative controls were stained by omission ofthe primary antibody.Two sections from each biopsy specimen

were stained and analysed for expression ofICAM-1, E-selectin, VCAM-1, and von

Willebrand factor. The numbers of immuno-reactive cells within the dermal infiltrate were

manually counted in six random fields ( x 40magnification, within a 1 mm2 grid) from twosections per biopsy specimen stained forCD11a, CD11b, CD11c, CD3, NP57, andCD68. The results were then averaged foreach group.The expressions of antigens (excluding

NP57 staining) were analysed separately usinganalysis of variance. The subjects were treatedas a random factor, the condition (control,lymphocytic, and leucocytoclastic vasculitis)as a between-subjects factor and the lesionstate (lesional or non-lesional) as a within-subjects factor. The residuals were checkedfor normal distribution using the Shapiro-Francia W test and the data were log trans-formed when necessary to produce normality.When log data were used, the differencebetween groups was presented as a ratio of thegeometric means. It was necessary to compareNP57 staining using the Mann-Whitney U

Table 2 Rrimary antibodies usedfor immunohistochemistry

Antibody Antigen Reference

6-5B5 ICAM-1 361-4C3 VCAM-1 361 2B6 E-selectin (ELAM-1) 36MHM24 CD1 la 37*44 CD1 lb 38t3 9 CD11c 39tA542 CD3 (pan T cell) 40*NP57 Neutrophil elastase 41*EBMI 1 CD68 (mononuclear phagocytes) 42*A082 Von Willebrand factor 43*

* Purchased from Dakopatts.tA kind gift from Dr Nancy Hogg, Imperial Cancer ResearchFund, London.

940

on July 24, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.10.939 on 1 O

ctober 1994. Dow

nloaded from

Adhesion molecule expression in vasculitis

Table 3 Numbers of cells stained positively for leucocyte integrins and cell markers inlesional and control biopsy specimens

Mean number ofimmunoreactive positive cells with 95% confidence intervals*

p Value(I) Leucocytoclastic (II) Lymphocytic (III) Control between

Antigen vasculitis (n = 4) vasculitis (n = 10) (n = 17) I and II

CDlla 114 (85-152) 213 (147-308) 11 (7-16) 001CD1lb 21 (11-39) 6 (3-13) 1 (0-3) 0-013CDI Ic 112 (76-166) 47 (28-79) 12 (6-21) 0 009CD3 44 (30-65) 242 (148-396) 18 (10-33) < 0 001CD68 113 (83-153) 106 (72-154) 27 (18-42) 0-79NP57 245 (165-311) 6 (1-12) 0 < 0 001

* Confidence intervals and p values were derived from analysis of variance on log transformeddata apart from NP57 which was compared using the Mann-Whitney U test and is thereforeexpressed as median values.t Included specimens from normal individuals and non-lesional skin from patients withvasculitis.

test due to the absence of staining in a largeproportion of biopsy specimens. The BMDPpackage (program 3V with REML option)was used to analyse the data.

ResultsThere was no difference in staining patternsfor each antibody between skin from normalsubjects and non-lesional skin specimens frompatients with vasculitis. These biopsy speci-mens are referred to collectively below as con-trol biopsy specimens.A significant increase in the numbers of

Table 4 Numbers of biopsy specimens stained positivelyfor ICAM-1, E-selectin, and VCAM-1 in lesional skin ofleucocytoclastic and lymphocytic vasculitis

Leucocytoclastic Lymphocyticvasculitis vasculitis

Keratinocyte ICAM-1 1 (7 0%) 8 (80%)Endothelial ICAM-1 14 (100%) 10 (100%)Endothelial E-selectin 8 (57%) 7 (70%)Endothelial VCAM-1 3 (21%) 5 (50%)

CD3 +, NP57 +, CD68 + and fl2 leucocyteintegrin positive cells was seen in all lesionalbiopsy specimens compared with those fromcontrols. Table 3 shows that there was anincrease in NP57 +, CD1 lb+, and CDllc+cells in leucocytoclastic compared with lym-phocytic vasculitis (p < 0-001, p = 0-013, p =0009, respectively), whereas CD11a+ andCD3+ cells were increased in lymphocyticvasculitis (p = 0 01, p < 0 001, respectively).There was no significant difference in thenumbers of CD68+ cells between the twovasculitic groups (p = 0 79).ICAM-1 was expressed constitutively on

endothelial cells in control biopsy specimens,precluding meaningful comparisons betweenlymphocytic and leucocytoclastic vasculitis.Keratinocyte ICAM-1 expression was absentin control biopsy specimens, and the propor-tion of specimens with detectable keratinocytestaining for ICAM-1 was significantlyincreased in lymphocytic vasculitis (eight of10, 80%) compared with leucocytoclastic vas-culitis (one of 14, 7%), p = 0-0013 (table 4).This ICAM-1 staining was almost exclusivelyconfined to basal keratinocytes (fig 1A) andwas seen close to the sites of CD 11 a (lympho-cyte function associated antigen-i) positivecells, either in the upper dermis or within theepidermis (fig 1B). Greater numbers ofICAM-1 positive dermal dendritic cells wereseen in lymphocytic vasculitis, although thenumber of positive biopsy specimens involvedwere too small for statistical comparisons.

E-selectin expression (figs 2A and B) wasnot present in control skin specimens but wasseen in postcapillary venules in both leucocy-toclastic (eight cases, 57%) and lymphocyticvasculitis (seven cases, 70%) (table 4).VCAM-1 expression, which was absent in

control biopsy specimens, was more fre-quently observed on endothelial cells in

- .~~~~~~~~"

"INN< }{ve0S~~rj4V ; f

(A) (B)

Figure 1 Lymphocytic vasculitis. (A) Strong ICAM-l expression confined to basal keratinocytes (immunoperoxidase);(B) same biopsy specimen as in fig IA showing CDllJa (lymphocyte function associated antigen-i) positive cells in theupper dermis and epidermis (immunoperoxidase).

941

on July 24, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.10.939 on 1 O

ctober 1994. Dow

nloaded from

Burrows, Molina, Terenghi, Clark, Haskard, Polak, Jones

Figure 2 E-selectin staining confined to vascular endothelial cells. (A) Leucocytoclastic vasculitis (immunoperoxidase);(B) lymphocytic vasculitis (immunoperoxidase).

lymphocytic (five cases, 50%) than leucocyto-clastic vasculitis (three cases, 21%), althoughthis was not clinically important (table 4). Aswith ICAM-1, VCAM-1 immunoreactivedermal dendritic cells were more pronouncedin lymphocytic compared with leucocytoclas-tic vasculitis, although the number of positivebiopsy specimens were too small for statisticalanalysis.

Reduced or absent staining of all threeendothelial adhesion molecules was seenwithin small areas of many lesional biopsyspecimens. This was associated with a diffuse,weak perivascular staining by von Willebrandfactor, suggesting antigen loss from theendothelial cytoplasm. These observationscorresponded to sites of pronounced fibrinoidnecrosis of blood vessels shown by haema-toxylin and eosin staining.

DiscussionThe pathophysiological mechanisms whichunderlie the development of cutaneous vas-culitic lesions are still poorly understood.Some of the events may be common toinflammatory responses in general, but othersare likely to determine the specific characteris-tics of a lesion. In this study we investigatedthe presence of three cell cytokine mediatedadhesion molecules in leucocytoclastic andlymphocytic vasculitis. We found that lym-phocytic vasculitis is associated, in most cases,with a pattern of adhesion molecule expres-sion that distinguishes the pathology of thislesion from that of leucocytoclastic vasculitis.

In contrast to lesional skin, we were unableto detect E-selectin or VCAM-1 expressionon blood vessels in biopsy specimens takenfrom non-lesional skin, indicating thatendothelial activation occurs in both forms of

vasculitis. Judging from in vitro experiments,the likely cytokines involved are either inter-leukin-1 or tumour necrosis factor,27 whichcould be released from a number of cells inthe vicinity of the blood vessel, includingmacrophages, lymphocytes, keratinocytes,smooth muscle cells, and endothelial cellsthemselves.3'

E-selectin was detected on endothelium oflesional skin in eight of 14 cases of leucocyto-clastic vasculitis and in seven of 10 cases oflymphocytic vasculitis. The absence of E-selectin staining in six of the leucocytoclasticbiopsy specimens may in part be related toendothelial injury as four showed pronouncedfibrinoid necrosis. The varying ages of the vas-culitic lesions are also likely to be important,although whenever possible early (less than 24hours' duration) lesions were biopsied. E-selectin is dependent on gene transcriptionand de novo protein synthesis and it is possiblethat the biopsy specimens might have beentaken at a time point preceding E-selectinexpression. More likely, however, E-selectinexpression may have been missed as it is seenmaximally two to four hours after cytokinestimulation.32

Endothelial VCAM-1 expression was seenin three of 14 cases of leucocytoclastic vasculi-tis and in five of 10 cases of lymphocytic vas-culitis. Although there was no differencebetween leucocytoclastic and lymphocyticvasculitis in the expression of VCAM-1 byendothelium, endothelial VCAM-1 expres-sion tended to be associated with greaternumbers of T cells in the tissues, implicatingVCAM-1 in lymphocyte recruitment.

Although both leucocytoclastic and lym-phocytic vasculitis were characterised byendothelial expression of E-selectin, the twoforms of lesion could largely be distinguished

942

on July 24, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.10.939 on 1 O

ctober 1994. Dow

nloaded from

Adhesion molecuk expression in vasculitis

by the presence or absence of ICAM-1expression by keratinocytes. Previous studieshave shown that expression of ICAM-1 bykeratinocytes is a feature of T lymphocytemediated responses2033 34 and is not aninevitable feature of cutaneous inflamma-tion.20 Our observations are therefore consis-tent with the lymphocytes emigrating into thetissues in lymphocytic vasculitis and playingan active part in the evolution of the lesions,perhaps by responding to antigen. Furtherstudies examining cell surface markers of Tcell activation and the presence of T cellderived cytokines, such as interferon y, whichare known to induce ICAM-1 expression bykeratinocytes,'435 should throw further lighton this matter.

Leucocytoclastic vasculitis, by definition,exhibits a perivascular neutrophil infiltrateand it was therefore anticipated that neu-trophil elastase (NP57), CD 1 lb, and CD 1cwould be found in the dermal infiltrate.Likewise, the inflammatory cells in lympho-cytic vasculitis stained positively for CD3 andCD1la. These results reflect cell lineage andno inference can be drawn from this studyregarding their role in cutaneous vasculitis.

In conclusion, this study has shown thatendothelial cell activation occurs in both leu-cocytoclastic and lymphocytic vasculitis, asdemonstrated by the presence of E-selectinexpression. Keratinocyte expression ofICAM-1 was also more common in lympho-cytic vasculitis, suggesting an active role for Tcells in the pathogenesis of this form of vas-culitic lesion.

NPB was supported by a grant from North West ThamesLocally Organised Research Scheme. FAM was in receipt of agrant of the programma nacional de becas de formaci6n depersonal investigador en el extranjero from the Ministerio deEducaci6n y Ciencia, Spain.

1 Braverman IM, Yen A. Demonstration of immune com-plexes in spontaneous and histamine-induced lesionsand in normal skin of patients with leukocytoclastic angi-itis. J Invest Dermatol 1975;64:105-12.

2 Gower RG, Sams WM, Thome EG, Kohler PF, ClamanHN. Leukocytoclastic vasculitis: sequential appearanceof immunoreactants and cellular changes in serial biop-sies. J Invest Dermatol 1977;69:477-84.

3 Smoller BR, McNutt NS, Contreras F. The natural historyof vasculitis. What the histology tells us about the patho-genesis. Arch Dermatol 1990;126:84-9.

4 Klug H, Haustein U-F. Ultrastructure of macrophage-lymphocyte interaction in purpura pigmentosa progres-siva. Dermatologica 1976;153:209-17.

5 Fauci AS, Haynes BF. The spectrum of vasculitis.Clinical, pathologic, immunologic, and therapeutic con-siderations. Ann Intern Med 1978;89:660-76.

6 Savel PH, Perroud A-M, Levy-Klotz B, Morel P. Vasculiteleucocytoclasique et lymphocytaire des petits vaisseauxcutanes. Ann Dermatol Venereol 1982;109:503-14.

7 Aiba S, Tagami H. Immunohistologic studies inSchamberg's disease. Evidence for cellular immune reac-tion in lesional skin. Arch Dermatol 1988;124:1058-62.

8 Simon M, Heese A, Gotz A. Immunopathological investi-gations in purpura pigmentosa chronica. Acta DermVenereol 1989;69:101-4.

9 Massa MC, Su WPD. Lymphocytic vasculitis: is it a spe-cific clinicopathological entity? J Cut Pathol 1984;11:132-9.

10 Zax RH, Hodge SJ, Callen JP. Cutaneous leukocytoclasticvasculitis. Serial histopathologic evaluation demon-strates the dynamic nature of the infiltrate. ArchDer,natol 1990;126:69-72.

11 Springer TA. Adhesion receptors of the immune system.Nature 1990;346:425-34.

12 Dustin ML, Rothlein R, Bhan AK, Dinarello CA, SpringerTA. Induction by IL 1 and interferon-y: tissue distribu-tion, biochemistry, and function of a natural adherencemolecule (ICAM-i). J Immunol 1986;137:245-54.

13 Dustin ML, Springer TA. Lymphocyte function-associ-ated antigen-I (LFA-1) interaction with inter-cellular

adhesion molecule-I (ICAM-1) is one of at least threemechanisms for lymphocyte adhesion to culturedendothelial cells. J CeU Biol 1988;107:321-31.

14 Dustin ML, Singer KH, Tuck DT, Springer TA.Adhesion of T lymphoblasts to epidermal keratinocytesis regulated by interferon-y and is mediated by intercellu-lar adhesion molecule I (ICAM-1). Jf Exp Med 1988;167:1323-40.

15 Smith CW, Rothlein R, Hughes B, Mariscalco M,Schmalstieg F, Anderson DC. Recognition of anendothelial determinant for CD 18-dependent neutrophiladherence and transendothelial migration. J Clin Invest1988;82: 1746-56.

16 Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S,Chi-Roso G, et al. Direct expression cloning of vascularcell adhesion molecule 1, a cytokine-induced endothelialprotein that binds to lymphocytes. Cell 1989;59:1203-11.

17 Rice GE, Bevilacqua MP. An inducible endothelial cellsurface glycoprotein mediates melanoma adhesion.Science 1989;246: 1303-6.

18 Thornhill MH, Haskard DO. IL-4 regulates cell activationby IL-1, tumour necrosis factor or IFN-y. J Immunol1990;145:865-72.

19 Swerlick RA, Lee KH, Li U, Sepp NT, Caughman SW,Lawley TJ. Regulation of vascular cell adhesion mole-cule 1 on human dermal microvascular endothelial cells.Jlmmunol 1992;149:698-705.

20 Norris PG, Poston RN, Thomas ST, Thornhill M, Hawk J,Haskard DO. The expression of endothelial adhesionmolecule-1 (ELAM-1), intercellular adhesion molecule-I(ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in experimental cutaneous inflammation: a compari-son of ultraviolet B erythema and delayed typehypersensitivity. J7 Invest Dermatol 199 1;96:763-70.

21 Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyj S,Hemler ME, et al. VCAM-1 on activated endotheliuminteracts with the leukocyte integrin VLA-4 at a site dis-tinct from the VLA-4/fibronectin binding site. Cell 1990;60:577-84.

22 Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS,Gimbrone MA. Identification of an inducible endothe-lial-leukocyte adhesion molecule. Proc Natl Acad SciUSA 1987;84:9238-42.

23 Picker U, Kishimoto TK, Smith CW, Wamock RA,Butcher EC. ELAM-1 is an adhesion molecule for skinhoming T cells. Nature 1991;349:796-9.

24 Shimizu Y, Shaw S, Graber N, Gopal TV, Horgan KJ, vanSeventer GA, et al. Activation-independent binding ofhuman memory T cells to adhesion molecule ELAM-1.Nature 1991;349:799-802.

25 Phillips ML, Nudeiman E, Gaeta FCA, Perez M, SinghalAK, Hakomori S-I, et al. ELAM-1 mediates cell adhe-sion by recognition of a carbohydrate ligand, Sialyl-Lex.Science 1990;250:1 130-2.

26 Berg EL, Yoshino T, Rott LS, Robinson MK, WamockRA, Kishimoto TK, et al. The cutaneous lymphocyteantigen is a skin lymphocyte homing receptor for thevascular lectin endothelial cell-leukocyte adhesion mole-cule 1._J Exp Med 1991;174:1461-6.

27 Pober JS, Bevilacqua MP, Mendrick DL, Lapierre LA,Fiers W, Gimbrone MA. Two distinct monokines: inter-leukin 1 and tumour necrosis factor, each independentlyinduce biosynthesis and transient expression of the sameantigen on the surface of cultured human vascularendothelial cells. Jf Immunol 1986;136:1680-7.

28 Copeman PWM, Ryan TJ. The problems of classification ofcutaneous angiitis with reference to histopathology andpathogenesis. BrJ Dermatol 1970;82(suppl 5):2-14.

29 Sternberger LA. The unlabelled antibody peroxidase-antiperoxidase (PAP) method. In: Immunocytochemistry.2nd edn. New York: John Wiley, 1979:104-69.

30 Shu S, Ju G, Fan L. The glucose oxidase-DAB-nickelenhancement method in peroxidase histochemistry ofthe nervous system. Neurosci Lett 1988;85:169-71.

31 Haskard DO. Cytokines, growth factors and interferons.In: LeRoy EC, ed. Systemic vasculitis. New York: MarcelDekker Inc, 1992:223-47.

32 Bevilacqua MP, Stengelin S, Gimbrone MA, Seed B.Endothelial leukocyte adhesion molecule 1: an induciblereceptor for neutrophils related to complement regula-tory proteins and lectins. Science 1989;243:1 160-5.

33 Lewis RE, Buchsbaum M, Whitaker D, Murphy GF.Intercellular adhesion molecule expression in the evolv-ing human cutaneous delayed hypersensitivity reaction.JInvest Dermatol 1989;93:672-7.

34 Singer KH, Tuck DT, Sampson HA, Hall RP. Epidernalkeratinocytes express the adhesion molecule intercellularadhesion molecule-1 in inflammatory dermatoses. JInvest Derinatol 1989;92:746-50.

35 Griffiths CE, Voorhees JJ, Nickoloff BJ. Characterizationof intercellular adhesion molecule-I and HLA-DRexpression in normal and inflamed skin: modulation byrecombinant gamma interferon and tumour necrosis_factor.'A Acad DeInatol 1989,20:617_29.

36 Wellicome SM, Thornmhil MH, Pitzalis C, Thomas DS,Lanchbury JSS, Panayi GS, et al. A monoclonal anti-body that detects a novel antigen on endothelial cellsthat is induced by tumour necrosis factor, IL-1, orlipopolysaccharide. J Immunol 1990;144:2558-65.

37 Hildreth JEK, Gotch FM, Hildreth PDK, McMichael AJ. Ahuman lymphocyte-associated antigen involved in cell-mediated lympholysis. Eury Immunol 1983;13:202-8.

38 Malhotra V, Hogg N, Sim RB. Ligand binding by the

943

on July 24, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.10.939 on 1 O

ctober 1994. Dow

nloaded from

Burrows, Molina, Terenghi, Clark, Haskard, Polak, Jones

pl50,95 antigen of U937 monocytic cells: properties incommon with complement receptor type 3 (CR3). Eur 7Immunol 1986;16:1117-23.

39 Hogg N, Takacs L, Palmer DG, Selvendran Y, Allen C.The pl50,95 molecule is a marker of human mononu-clear phagocytes: comparison with expression of class IImolecules. EurJ Immunol 1986;16:240-8.

40 Erber WN, Pinching AJ, Mason DY. Immuno-cytochemical detection of T cell and B cell populationsin routine blood smears. Lancer 1984;i:1042-5.

41 Pulford KAF, Erber WN, Crick JA, Olsson I, Micklem KJ,Gatter KC, et al. Use of monoclonal antibody againstneutrophil elastase in normal and leukaemic myeloidcells. Clin Pathol 1988;41:853-60.

42 Kelly PMA, Bliss E, Morton JA, Burns J, McGee JO'D.Monoclonal antibody EBM/I 1: high cellular specificityfor human macrophages. Clin Pathol 1988;41:510-5.

43 Sehested M, Hou-Jensen K. Factor VII-related antigenas an endothelial cell marker in benign and malignantdiseases. Virchows Arch (PatholAnat) 1981;391:217-25.

944

on July 24, 2021 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.10.939 on 1 O

ctober 1994. Dow

nloaded from