Annals of the Rheumatic Diseases 1995; 54: 209-215

Cytokine and adhesion molecule expression in theminor salivary glands of patients with Sjogren'ssyndrome and chronic sialoadenitis

Alberto Cauli, Ghada Yanni, Costantino Pitzalis, Steven Challacombe, Gabriel S Panayi

AbstractObjective-To investigate the role ofcytokines and cell adhesion molecules inthe pathogenesis of Sjogren's syndrome(SS).Methods-Using an indirect immuno-peroxidase technique we assessed theexpression of the cytokines interleukin-Ia (IL-la), interleukin-i 0 (IL-1), inter-leukin-8 (IL-8), transforming growthfactor B (TGFO) and granulocyte macro-phage colony stimulating factor (GM-CSF),of the adhesion molecules intercellularadhesion molecule-i (ICAM-1), lympho-cyte function associated antigen-I (LFA-1),the activated molecular form of LFA-1(NKI-Li6), CD2, and LFA-3, and of apanel of cellular markers in the minorsalivary glands.Results-In SS and chronic sialoadenitis(CS), the ductal epithelial cells and aciniexpressed all the cytokines examined. Thepercentage of glandular mononuclearcells which stained positive for cytokinesdid not differ significantly between SSand CS. NKI-L16 was detected on33-6 (SD 10i1)% and 15-3 (4.3)% of LFA-1cells in SS and CS, respectively(p < 0.002).Conclusion-SS and CS did not differ inthe pattern of cytokines examined. Thecharacteristic cell clustering seen in thesalivary glands in SS may be caused bythe upregulation ofNKI-L16.

(Ann Rheum Dis 1995; 54: 209-215)Rheumatology Unit,Division ofMedicine,United Medical andDental Schools,Guy's Hospital,London,United KingdomA CauliG YanniC PitzalisG S PanayiDepartment of OralMedicine andPathologyS ChallacombeCorrespondence to:Professor G S Panayi,Rheumatology Unit,Department of Medicine,Fourth floor,Hunt's House,Guy's Hospital,St Thomas Street,London SE1 9RT,United Kingdom.Accepted for publication14 October 1994

Sjogren's syndrome (SS) is an autoimmunedisease characterised by a periductal mono-nuclear cell (MNC) infiltrate resulting indistinct focal cellular aggregates of the salivaryand lachrymal glands, leading to a chronicinflammatory process with secondary symp-toms and signs of dry mouth and dry eyes andby the production of autoantibodies.' 3 Theinfiltrating cells are predominantly CD4 Tcells, but plasma cells and macrophages are

also present.45 Despite extensive histological,immunopathological and virological studies,the triggering factors responsible for theautoimmune lesion in the glandular tissue ofSS remain obscure.

Cytokines have been implicated in thepathogenesis of many inflammatory lesions.Previous studies, applying immunohisto-chemical techniques to the salivary glands of

patients with SS, demonstrated that interferongamma (IFN'y) was extensively expressed inthe acini, ducts, and MNCs, but only 244%of infiltrating cells expressed interleukin-2(IL-2).6 7 This was further confirmed by in situhybridisation and the reverse transcriptasepolymerase chain reaction (RT-PCR),8 '0 inwhich mRNA for IFNy and IL-2 was detectedin the salivry glands of all patients studied,while interleukin-4 (IL-4) was absent.8 Theseobservations suggest continuing T cellactivation ofthe ThI cell type in the SS lesions,as reflected by the local production of IL-2 andIFN-y. Furthermore, biopsies of salivary glandsfrom SS patients with extensive lymphoidinfiltrates contained mRNA for IL-2 andIFNy, while biopsies with few lymphoidinfiltrates had large amounts of transforminggrowth factor ( (TGFI3.)° It was suggested,therefore, that the greater concentrations ofTGF,3 in minimal lesions may play a role indownregulating the inflammatory process.Support for this hypothesis comes from amouse model in which disruption of the TGF(gene by homologous recombination in murineembryonic stem cells makes the animals unableto mount an efficient anti-inflammatoryresponse. The salivary glands of these micewere characterised by a periductal lympho-plasmacytic infiltrate not unlike that associatedwith SS."

Chronic sialoadenitis (CS) is a non-specificinflammatory process of the salivary glandscharacterised by a sparse diffuse MNCinfiltrate in the gland leading to glandularatrophy with secondary signs and symptomsof dry mouth, but not the presence ofautoantibodies. Thus this disease is the idealcontrol for immunopathological studies in SS.We decided, therefore, in the first instance toinvestigate the expression of cytokines suchas TGF(, IL-lit, IL-1i, IL-8 and granulo-cyte macrophage colony stimulating factor(GM-CSF) in SS and CS to see if these twoconditions could be distinguished. We alsoexamined salivary glands from five healthynormal control subjects. In order to clarifythe typically different (focal versus diffuse)distribution and organisation of the MNCinfiltrate in SS and CS, we then studied theexpression of specific adhesion molecules(lymphocyte function associated antigen-I(LFA-1)/intercellular adhesion molecule-1(ICAM-1) and CD2/LFA-3) involved inhomo- and heterotypic cluster formation.Finally, we examined the expression of anactivation dependent epitope of the LFA- 1

209

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from

Cauli, Yanni, Pitzalis, Challacombe, Panayi

molecule recognised by the monoclonalantibody (MAb) NKI-L16.12

Patients and methodsPATIENTS

We studied six patients with definite SS (fourprimary and two secondary with rheumatoidarthritis; mean age 64-6 (range 40-79 years; allfemale) who fulfilled the American College ofRheumatologists criteria and European criteriafor SS.2 3 This group was characterised bysymptoms and signs of ocular and oralinvolvement, and the presence of antinuclearantibodies, and SS-A and SS-B antibodies.Four of five patients tested were rheumatoidfactor positive and all demonstrated a typicalperiductal infiltrate on histological analysis ofthe minor salivary glands. We also studied sixpatients with CS (mean age 58-3 range 57-76years; five females and one male) with siccasyndrome and the histopathological featuresof CS. This group was characterised byoral involvement (xerostomia), no signs orsymptoms of lachrymal involvement, absenceof autoantibodies (rheumatoid factor, SS-Aand SS-B) and the presence of a lymphocyticinfiltrate in the salivary glands. Five biopsiesfrom healthy volunteers were also studied.

TISSUE PREPARATION AND STAINING

Biopsies containing at least two foci per 4 mm2were obtained from minor salivary glands fromthe midline of the lower lip and embeddedin optimal temperature cutting compound(OCT, Miles, CA) and snap frozen inisopentane and liquid nitrogen. Samples werestored at -70°C until sectioned for immuno-histological staining. Sections 5 ,um thick werecut with a cryostat (Leitz) at -22°C. Sequentialsections were mounted on poly-L-lysine coatedslides and dried overnight at room tem-perature. Sections were fixed in acetone for 10minutes, wrapped in tin foil, and stored at-70°C until further use.

Cytokines and cellular antigens weredetermined by an indirect immunoperoxidasetechnique. The MAbs used to determinecellular content were OKT3, OKT4, OKT8,(anti-CD3, anti-CD4, anti-CD8, respectively;ATCC, USA), B-Lyl, and EBMI 1 (anti-CD20, a B cell marker and anti-CD68, amacrophage marker, respectively; Dakolaboratories, Copenhagen, Denmark). Anti-factor VIII related antigen (Dako) was used tostain endothelial cells. The MAbs used todetermine cytokine expression were anti-IL-la(gift of Dr R Thorpe, NIBSC, Potters Bar),anti-IL- 1, (gift of Dr D Boraschi, Italy), anti-GM-CSF (gift of Dr K Ruedi, Sandoz, Basel,Switzerland), anti-IL-8 (gift of Dr M Ceska,Vienna) and anti-TGFI (gift of Dr Feldman,Genentech, USA). The MAbs used to detectthe adhesion molecules were anti-LFA- 1 (TS 1/22hybridoma clone, gift ofDr T Springer, USA),NKI-L1 6 (gift of Prof Figdor, Netherland),anti-ICAM-1 (gift of Dr D Haskard, London,UK), anti-CD2 and anti-LFA3 (BectonDickinson, CA, USA).

Briefly, the salivary gland tissue sections wereincubated for 10 minutes with a 1:20 dilutionofnormal rabbit serum in a humidified chamberat room temperature. The sections were thenincubated with the primary MAbs, at appropri-ate dilutions in phosphate buffer saline (PBS)for two hours. The excess MAb was removed bywashing with PBS. The second antibody, horse-radish peroxidase conjugated rabbit anti-mouse(Dako laboratories, Copenhagen, Denmark)diluted in PBS at 1: 100, was added and sectionswere incubated for 30 minutes. After rinsingwith PBS the sections were developed in asolution of diaminobenzidine tetrahydro-chloride 0 7 mg/ml, and counterstained withhaematoxylin. Finally, sections were dehydratedby transferring through alcohol and CNP30,and mounted in DPX.

Negative control staining was performed onall salivary gland specimens studied, followingan identical procedure except that incubationwith the primary MAb was omitted; no stainingwas noted in these sections. Tonsil tissue wasused as a positive control. Other controls in-cluded MOPC21-a non-immune mouse IgGImyeloma protein MOPC2 1, with an isotypesimilar to the cytokine MAbs used, which wasused at equivalent protein concentration.

All comparisons between specimens weremade with data derived from the same stainingrun.

CONFIRMATION OF CYTOKINE

IMMUNOREACTIVITYThe specificity of the cytokine MAbs has beenextensively studied'3 and demonstrated by theproviders of these reagents (IL-la,14 IL-i ,15GM-CSF,'6 IL-817 and TGFI3'8). The speci-ficity of the binding of these antibodies tosalivary gland tissue was confirmed by: stainingwith cytokine MAb (staining was concentra-tion dependent); using an irrelevant antibody,MOPC2 1, at equivalent protein concentration;comparing the pattern and degree of stainingof different MAbs for the same cytokineproduct on salivary gland and tonsil whichyielded the same pattern of staining; and veri-fying the increased cytokine production byphytohaemagglutinin stimulated versus restingperipheral blood MNCs.

MICROSCOPIC EVALUATION

All microscopic evaluations were performed ina coded fashion by one observer (AC) who wasblinded to the name of the patients anddiagnosis. To rule out intra-observer variation,the samples were randomised, recoded, andread on a second occasion. (No significantdifferences were demonstrated between thetwo readings.) Samples were also assessed byanother investigator (GY) to estimate inter-observer variation. (No significant differenceswere found between the two investigators.)

NUMBER OF INFILTRATING CELLS

For each patient, the total number ofinfiltrating cells was estimated by counting all

210

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from

Cytokine and adhesion molecule expression in patients with Sjdgren's syndrome and chronic sialoadenitis

the cells throughout the minor gland biopsiedand expressing the results per mm2.

50

iTT40

C',

00

0 L

CD3 CD4 CD8 CD20 CD68

Figure 1 Phenotypic analysis of the mononuclear cellular infiltrate in Sjdgren syndrome(U) and chronic sialoadenitis (LI).

CELL SUBSETSThe number of cells staining positively for aparticular cell marker was counted in a total of500 cells, and the results expressed as apercentage.

CYTOKINE STAINING

Cytokine expression was assessed in three areasof the salivary glands: infiltrating cells, ducts,and acini. In the infiltrate, cytokine stainingwas assessed as a percentage of positivelystaining cells in a total of 500 cells, while in theacini and ducts the intensity of staining wasestimated by devising a scale of 0-8, where0 = no staining and 8 = maximal staining.

VASCULAR PROLIFERATION

Vascular proliferation was estimated bycounting the number of blood vessels stainingwith factor VIII and expressing the resultsper mm'.

@.- 4td, _* i , g;

;zz t,:44" ~~';-:r4Xv4¶.14,_f

a

/

X.40..,# .-14

I

., [e.X-'%t*

-.0 I"

:-.9..C 0 MP

X

I1

I49

IVibdi

IT.-_',.

.1

Ai

.ftm ,4

A1

Figure 2 Interleukin-8 staining in Sjogren 's syndrome (A), chronic sialoadenitis (B), and normal controls (C).Transforming growth factor beta staining in Sjigren's syndrome (D), chronic sialoadenitis (E), and normal controls.A, B, D, E: 10 mm represents 34-5 ,m; C, F: 10 mm represents 19-5 ,tm.

60

9o

4

4:.

.C.

21

':a X

*

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from

Cauli, YaTiho, Pitzalis, Challacombe, Panacvi

ADHESION MOLECULES

Adhesion molecule expression was assessed inthe three areas of all salivary gland tissuesections and their distribution analysed. Forthe expression of NKI-L16, the number ofNKI-L16 positive cells in a total number of500 LFA-1 positive cells was counted and theresults expressed as a percentage.

60-T)

o50

400

30

20-

10

0

IL-1 IL-~1 TGF[ GM-CSF

Figure 3 Percentage of staining cells for the cytokines interleukin- I a (IL-I K)interleukin-I (IL- /3,), transforning growth factor /3 (TGF,3) and granulocyteitnacrophage colony stimulating factor (GM-CSF) in the mononuclear cellularinfiltrate of the minor salivarv glands ofpati'ents with Sjogren 's syndrome (-) andchronic sialoadenitis (LI).

STATISI1 ICS

Results are expressed as mean (SD). Histo-logical scores in the three different groups werecompared using the Mann-Whitney test.

ResultsHISTOI.OGIC,AL FEATURES AND CELLULAR

COMPOSITION

Minor salivary gland biopsies from patientswith SS demonstrated typical focal lympho-cytic clusters of more than 50 cells per focus.In contrast, in CS the infiltrating cells wererandomly scattered throughout the gland. Noinfiltrating cells were observed in normalcontrols. Significantly greater numbers ofinfiltrating cells per mm' of tissue wereseen in SS (1717 1 (1083 3) cells/mm2) com_pared with CS (537 0 (584 2) cells/mm2)(p < 0 031). Phenotypic analysis of the infil-trating cells in the two pathological groupsrevealed no statistically significant differ-ences (fig 1) It is worth noting however,that the CD4:CD8 ratio was greater in SS(2 8) compared with CS (1 8), reflecting thegreater number of CD4 cells seen in SS(33 6 (16 4) cells/mm2) compared with CS(21 2 (13 5) cells/mm2) The number of CD8cells was similar in the two groups (14 6 (10 3)and 16-6 (12 1) cells/mm2, respectively).Greater numbers of CD20 cells were countedin SS (20 0 (9 5) cells/mm2) than in CS(13 3 8 9) cells/mm2). There was a tendencyto greater numbers of CD68 cells in CS(16 2 (10 9) cells/mm2) compared with SS(8-0 (5 4) cells/mm2). The number of bloodvessels per mm of tissue in SS (134 1 (2118)vessels/mm') was not significantly differentfrom that in CS (129-5 (10-4) vessels/mm2),but in both diseases more blood vessels werepresent compared with normal subjects(100 5 (10-8) vessels/mm2) (p= 0015 andp = 0 004 versus SS and CS, respectively).

C:c

0

l_

U)

c

a)c

IL-1 IL-1. IL-8 GM-CSF TGFf

Figure 4 Intensity of staining of the cvtokines inlterleukin-1 a (IL-l as) intterlelukin-i /8(IL-1,B), interleukin-8 (IL-8), granulocyte miiacrophage colony stimnulatingfactor(GM-CSF) and transforming growth factor ,3 (TGF/,) in the ducts of the minor

salivary glands ofpatients with Sjigren 's svyndromne (-) or chronic sialoadenitis (LI),and nornial controls (n,).

CYTl-OKINE EXPRESSION

Mononuclear cellular infiltrate. IL-i x, IL-i,

TGF[ and GM-CSF were expressed in theMNC infiltrate of both SS and CS (fig 2). Nostatistically significant differences were notedin the percentages of positively staining cells in

the two patient groups (fig 3). IL-8 expressionwas minimal (<2%) in the MNC infiltrate ofall minor salivary glands examined.

Ducts. All the cytokines studied were

strongly expressed in the epithelial cells of theducts in SS and CS, but at a much lower levelin normal controls. As can be seen in figure 4,the intensity of cytokine staining was sig-nificantly greater in the disease groups

compared with control subjects, except forGM-CSF. No statistically significant differ-ences were noted in the intensity of stainingbetween SS and CS. There was no correlationbetween the intensity of ductal cytokinestaining and the number of infiltrating cellssurrounding the same duct (data not shown).

Acini. All cytokines studied were expressedin the acini of SS, CS, and normal controls.The intensity of cytokine staining in the acini

100 -

90 -

80 -

70 -

212

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from

Cytokine and adhesion molecule expression in patients with Sjogren's syndrome and chronic sialoadenitis

7.

6-

4)W

4n0

0C

5-

4-

3'

0 I NEEL- rIZ MOM V_IL-la IL-15 IL-8 GM-CSF

Figure 5 Intensity ofstaining of the cytokines interleukin-1 a (IL-la) interleuk(IL-i,8), interleukin-8 (IL-8), granulocyte macrophage colony stimulatingfactoi(GM-CSF) and transforming growth factor (3 (TGF,B) in the acini of the minorsalivary glands ofpatients with Sjogren's syndrome (U) or chronic sialoadenitisand normal controls (F).

was significantly less than in the distatistically significant differences wein the cytokine staining between SSThe intensity of staining of somecytokines examined was greater in SScompared with controls (fig 5).

Vascular. IL-lot, IL-i1 and TGF,Bwas noted in the majority of the bloocbut GM-CSF and IL-8 were expresminority of vessels (data not shown).

ADHESION MOLECULES

The adhesion molecules examined i

tected at a similar level in both diseas4

LFA-3 was expressed by acini, ducts, bloodvessels, and MNC in both SS and CS. ICAM-1 was equally detected on blood vessels andMNC of both diseases. Likewise, LFA-1 andCD2, found on MNC, were equally expressedin SS and CS. However, there was a strikingdifference in expression of NKI-L16, whichwas found on 33-6 (10-1)% and 15-3 (4 3)%of LFA-1 positive cells in SS and CS,respectively (p < 0002) (fig 6).

DiscussionIn this study, analysis of cytokine expressionby the infiltrating MNC revealed a similarpercentage of IL-lot, IL-1,B, IL-8, GM-CSFand TGF,B positively staining cells in Sjogren'ssyndrome and chronic sialoadenitis. In bothdisease groups, the ducts and acini showedmore intense staining of all cytokines examinedcompared with normal control subjects. No

TGFO3 significant difference was noted between SSand CS. Acinar staining was less intense than

un-il p ductal staining, supporting the previoussuggestion of a minor role for the acinar

([1), epithelial cells compared with ductal epithelialcells in the salivary gland inflammatory processas shown by the expression of class II

acts. No molecules in the epithelial cells. 19-21 There noted HLA-DR upregulation may be caused byand CS. IFN-y produced locally at the site ofof the inflammation, as it is supported by theand CS reported increase in IFNy staining in SS

compared with controls.7 The aberrantstaining expression of class II molecules could be

d vessels, related to the potential antigen presenting,sed in a function of epithelial cells, as described in

the epithelial cells from gut and respiratorymucosae, epidermis, and renal proximaltubules.22 24The strong expression of TGF,B which we

were de- observed in the epithelial, acinar, and in-e groups. filtrating cells clearly demonstrates that in SS

;.VF::4

,AU

I

rC)

.4 .1 - , 146.,&,O-..,j ,A f or

.v ./flb .

J:.-.,.f

I., ,;,A.r,4

41.*. g s ; F ~~~~~~~A -jj

Jrfl*Jo~tZ_@'* r#S t *

4

V

4. -

, . 7 4.

F. .

A.-'U',"

-p

1A

aSU,{

"-a _..

Figure 6 Staining oflymphocyte function associated antigen-1 (LFA-1) (A) and NKI-L16 (B) in the minor salivaryglands ofSjogren's syndrome. Staining ofLFA-1 (C) and NKI-L16 (D) in the minor salivary glands of chronicsialoadenitis. A-D: 10 mm represents 26-6 Am.

rb .

213

f, .0

0. "

'.

t - , *... 11-.91 .4 1. -.I

G. ;.

e

t

.1.

I

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from

Cauli, Yanni, Pitzalis, Challacombe, Panayi

there is no obvious TGF, deficiency, but theactivation state of TGFP needs to bedetermined before any firm conclusions can bemade. We did not demonstrate a correlationbetween the intensity of cytokine ductalstaining and the number of surroundingMNCs. Our findings do not support otherworkers' observations of high expression ofTGFP in salivary glands of patients with SSwith a minimal infiltrate compared with thosewith an intense infiltrate.'0 It is possible that anunknown cytokine or other inflammatorymediator not examined in this work may beresponsible for the periductal clustering of theinfiltrating cells so typical in SS. Furthermore,it is necessary to study agonists and antagonistssimultaneously, as the balance between thesetwo may determine the nature of the disease.Although there were greater numbers of

CD4 and CD20 cells in SS compared withCS, the difference did not reach statisticalsignificance. However, the number of MNCthroughout the SS salivary glands was threetimes greater. The organisation of MNC intofoci in SS and sparsely diffuse infiltrates in CSwas a striking difference between the twogroups. In chronically inflamed non-lymphoidtissues, infiltrating lymphocytes form aggre-gates which are functionally and structurallysimilar to follicles found in lymph nodes ortonsil. The presence of focal lymphoidaggregates in the rheumatoid synovium isassociated with greater production of cytokineby the synovial membrane.25 In this study,we confirmed the presence of similar largelymphocyte aggregates in the salivary glands inSS, but were not able to show greater cytokinestaining in the aggregates seen in SS.The histological differences noted between

SS and CS cannot be attributable to a greaterdegree of vascularisation in SS, as the numbersof blood vessels in SS and CS were similar.However, the differences may lie in theexpression of adhesion molecules: we demon-strated a striking increase in the percentage ofNKI-L16/LFA-1 cells in SS compared withCS. Cell adhesion by LFA-1 is established bythe binding of LFA-1 to its ligands (ICAM-1and ICAM-2) on the opposing cell26 27 resultingin homotypic and heterotypic aggregation.In this study, salivary gland expression ofNKI-L16, the activated LFA-1 epitope, corre-lated well with the capacity of cells to aggregateand to form the characteristic foci seen in thesalivary glands of patients with SS. A pre-ponderance of memory cells with CD45ROmarker has been shown in SS salivary glands;28these cells have been reported to adhere betterto endothelial cells and to form large homo-typic cell clusters compared with CD45ROnegative cells.29 It has been suggested thatthe LFA-1/ICAM-1 and vascular leucocyteantigen-4 (VLA-4)NCAM-1 pathways mightbe the main routes for T cells to migrate to sitesof salivary gland inflammation in SS.28 Thissuggestion is supported by the extensivecoexpression of LFA- 1 or VLA-4 andCD45RO by CD4 T cell infiltrates adjacent toICAM-1 or VCAM-1 postcapillary venules.These findings may point to a cytokine

mediated upregulation of ICAM- 1 andVCAM- 1, facilitating the recruitment ofLFA-1/NKI-L16 and VLA-4 cells andresulting in the infiltration of the salivaryglands in SS.

In conclusion, this study shows that SS ischaracterised by periductal clustering ofMNC,while CS is characterised by a sparse infiltrate.No differences were noted in the subset ofinfiltrating MNC and cytokine expression inthe salivary glands of the two patient groups,but there was upregulation of NKI-L16 in theMNC of SS, suggesting that the typical MNCaggregation seen in SS may be mediated byactivation of LFA- 1.

This study was supported by a core grant (U9) from theArthritis and Rheumatism Council of Great Britain.

1 Fox R I, Howell F V, Bone R C, Michelson P E. PrimarySjogren's syndrome: clinical and immunopathologicfeatures. Semin Arthritis Rheum 1984; 14: 77-105.

2 Fox R I, Robinson C A, Curd J G, Kozin F, Howell F V.Sjogren's syndrome: proposed criteria for classification.Arthritis Rheum 1986; 29: 577-85.

3 Vitali C, Bombardieri S, Moutsopoulos H M et al.Preliminary criteria for the classification of Sjogren'ssyndrome. Arthritis Rheum 1993; 36: 340-7.

4 Talal N, Silvester R, Daniels T, Greenspan J, Williams R.T and B lymphocytes in peripheral blood and tissuelesions in Sjogren's syndrome. J Clin Invest 1974; 53:180-9.

5 Adamson T C, Fox R I, Frisman D M, Howell F V.Immunohistologic analysis of lymphoid infiltrates inprimary Sjogren's syndrome using monoclonal anti-bodies. Jl Immunol 1983; 130: 203-8.

6 Fox R I, Theofilopoulos A N, Altman A. Production ofinterleukin 2 (IL 2) by salivary gland lymphocytes inSjogren's syndrome. Detection of reactive cells by usingantibody directed to synthetic peptides of IL 2. J Immunol1985;135:3109-15.

7 Rowe D, Griffiths M, Stewart J, Novick D, Beverley P CL, Isenberg D A. HLA class I and II, interferon,interleukin 2, and the interleukin 2 receptor expression onlabial biopsy specimens from patients with Sjogren'ssyndrome. Ann Rheum Dis 1987; 46: 580-6.

8 Skopouli F N, Boumba D, Moutsopoulos H M. CytokinemRNA expression in the labial salivary gland tissue frompatients with primary Sjogren's syndrome. Clin Rheumatol1993; 12: 18.

9 Fox R I, Ando D, Abrams J, Kang H I. Characterizationof cytokine mRNA transcripts in salivary gland biopsiesfrom patients with Sjogren's syndrome (SS). ArthritisRheum 1992; 35: S122.

10 Ogawa N, McGuff H, Dang H, Aufdermorte T, Talal N.Cytokine profiles of salivary glands from patients withSjogren syndrome. Arthritis Rheum 1993; 36: S43.

11 Shull M M, Orrmsby I, Kier A B, et al. Targeted disruptionof the mouse transforming growth factor-, 1 gene results inmultifocal inflammatory disease. Nature 1992; 359: 693-9.

12 van Kooyk Y, Weder P, Hogervost F, et al. Activation ofLFA-1 through a Ca"+ dependent epitope stimulateslymphocyte adhesion. J Cell Biol 1991; 112: 345-54.

13 Farahat M N, Yanni G, Poston R, Panayi G S. Cytokineexpression in synovial membranes of patients withrheumatoid and osteoarthritis. Ann Rheum Dis 1993; 52:870-5.

14 Thorpe R, Wadh W A, Glearing A J H, Mahon B,Poole S. Sensitive specific immunoradiometric assay forhuman interleukin alpha. Lymphokine Res 1988; 7:119-24.

15 Boraschi D, Volpini G, Villa L, et al. A monoclonal antibodyto the Interleukin-I beta peptide 163-171 blocksadjuvanticity but not pyrogenicity of IL- lb in vivo.lImmunol 1989; 143: 131-4.

16 Zenke G, Strittmatter U, Tees R, Andersen E, Fagg B,Kocher H, Schreier M A. Cocktail of three monoclonalantibodies significantly increases the sensitivity of anenzyme immunoassay for human granulocyte macro-phage colony stimulating factor. J Immunol 1991; 12:185-206.

17 Ceska M. Interleukin-8 immunoreactivity in the skin ofhealthy subjects and patients with palmoplantarpustulosis and psoriasis. J Invest Dermatol 1992; 98:96-101.

18 Lucas C, Bold L N, Fendly B M, et al. The auto-crine production of transforming growth factor betaduring lymphocyte activation. A study with a mono-clonal antibody-based ELISA. J Immunol 1990; 145:1415-22.

19 Speight P M, Cruchley A, Williams D M. Epithelial HLA-DR expression in labial salivary glands in Sj6gren'ssyndrome and non-specific sialoadenitis. J Oral PatholMed 1989; 18: 178-83.

214

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from

Cytokine and adhesion molecule expression in patients with Sjdgren 's syndrome and chronic sialoadenitis

20 Fox R I, Bumol T, Fantozzi R, Bone R, Schreiber R.Expression of histocompatibility antigen HIA-DR bysalivary gland epithelial cells in Sjogren's syndrome.Arthritis Rheum 1986; 29: 1105-11.

21 Thrane P S, Halstensen T S, Haanaes H R, Brandt-zaeg P. Increased epithelial expression of HLA-DQ andHLA-DP molecules in the salivary glands frompatients with Sj6gren's syndrome compared with ob-structive sialoadenitis. Clin Exp Immunol 1993; 92:256-62.

22 Mayer L, Shlien R. Evidence for function of la moleculeson gut epithelial cells in man. JT Exp Med 1987; 166:1471-83.

23 Grabbe S, Bruvers S, Gallo R L, Knisely T L, Nazareno R,Granstein R D. Tumor antigen presentation by murineepidermal cells. _l Immunol 1991; 146: 3656-61.

24 Hagerty D T, Allen P M. Processing and presentation of selfand foreign antigens by the renal proximal tubule.JIImmunol 1992; 148: 2324-30.

25 Yanni G, Whelan A, Feighery C, Symons J, Duff G,Bresnihan B. Contrasting levels of in vitro cytokineproduction by rheumatoid synovial tissue demonstratingdifferent pattems of mononuclear cell infiltration. ClinExp Immunol 1993; 93: 387-95.

26 Marlin S D, Springer T A, Purified intercellular adhesionmolecule-I (ICAM-1) is a ligand for lymphocyte function-associated antigen-i (LFA-1). Cell 1987; 51: 813-9.

27 Staunton D E, Dustin M L, Springer T A. Functionalcloning of ICAM-2, a cell adhesion ligand for LFA-1homologous to ICAM-1. Nature 1989; 339: 61-4.

28 Saito I, Terauchi K, Shimuta M, et al. Expression of cell-adhesion molecules in the salivary and lacrimal glands ofSj6gren's syndrome.JClin Lab Analysis 1993; 7: 180-7.

29 Pitzalis C, Kingsley G H, Haskard D, Panayi G S. Thepreferential accumulation of helper-inducer T lympho-cytes in inflammatory lesions: evidence for the regulationby selective endothelial and homotypic adhesion. EurImmunol 1988; 18: 1397-404.

215

on January 10, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.54.3.209 on 1 M

arch 1995. Dow

nloaded from