Thorax 1993;48:1140-1144

Intercellular adhesion molecule 1 (ICAM-1) inthe pathogenesis of mononuclear cell alveolitis inpulmonary sarcoidosisKlaus Dalhoff, Sabine Bohnet, Jorg Braun, Burkhard Kreft, Karl J Wief3mann

Department ofInternal Medicine,Medical University ofLfbeck, RatzeburgerAllee 160, D-23538Lubeck, GermanyK DalhoffS BohnetJ BraunB KreftK J WieB3mannReprint requests to:Dr med K DalhoffReceived 16 November 1992Returned to authors3 March 1993Revised version received13 April 1993Accepted 11 August 1993

AbstractBackground-Alveolitis in pulmonarysarcoidosis is characterised by anaccumulation of highly activatedmacrophages and CD4+ lymphocytes inthe alveolar compartment. The role ofintercellular adhesion molecule 1(ICAM-1) expression on alveolar cellshas been studied in this context.Methods-Using a sandwich ELISAtechnique, ICAM-1 expression on alveo-lar macrophages from 17 consecutiveuntreated patients with pulmonarysarcoidosis and six healthy normalvolunteers was quantified. In addition,parameters of macrophage activation(tumour necrosis factor a (TNFa) andsuperoxide anion release) wereevaluated.Results-Significantly elevated expres-sion could be demonstrated on alveolarmacrophages from patients withpulmonary sarcoidosis compared withhealthy controls (mean (SD) 0-74 (0.24)ELISA units (EU) v 0-46 (0-12) EU). Onsubdividing the patients into those withactive and those with inactive disease,only the former showed increasedICAM-1 levels on alveolar macrophages(0.82 (0.27) EU) compared with controlalveolar macrophages. No differenceswere detected in serum levels of solubleICAM-1 between patients and controls.ICAM-1 expression on alveolar macro-phages from patients with sarcoidosiscorrelated with the spontaneous releaseof TNFa but not with the release of thesuperoxide anion by the activatedmacrophages. There was no correlationwith the percentage of lymphocytes orthe absolute number of CD4+ cells inbronchoalveolar lavage fluid.Conclusions-Increased ICAM-1 surfaceexpression on alveolar macrophagesreflects disease activity in the pulmonarycompartment. Considering the signifi-cance of adhesion molecules duringantigen presentation and lymphocyteactivation, ICAM-1 expression onalveolar macrophages may have animportant role in the immune process ofpulmonary sarcoidosis.

(Thorax 1993;48:1140-1144)

Leucocyte homing to sites of acute or chronicinflammation is a crucial step during theinflammatory response. Adhesion moleculessuch as intercellular adhesion molecule 1(ICAM-1) play a major part in this process bymediating adherence of leucocytes to theendothelium and initiating the extravasationof those cells.' At the site of inflammation theinteraction of ICAM-1 (CD54) with itsligands LFA-1 (CD1 la/CD 1 8) and Mac-i(CD1 lb/CD 18) provides an antigen indepen-dent mechanism of cell-cell activation inaddition to the antigen dependent interactionmediated by molecules of the MHC class IIcomplex and the T cell receptor.2 Undernormal conditions the f2 integrins, which arerestricted to leucocytes, exist in an inactiveform. They are activated rapidly by a numberof substances including lipopolysaccharide(LPS) and phorbol-12-myristate-1 3-acetate(PMA).3 In contrast, ICAM-1 is not normallypresent at the cell surface but can be inducedwithin hours during inflammation." Signalswhich are transduced at least partially by theICAM-1/fl, integrin pathway include lympho-cyte proliferation, antigen processing, andphagocytosis.7

Pulmonary sarcoidosis represents anexample of compartmentalised inflammation,with accumulation of activated macrophagesand CD4+ lymphocytes in the lung.-10Central effector mechanisms of primedmacrophages include the release of cytokines,reactive oxygen species, and proteases.11-13However, the mechanisms leading to localupregulation of the inflammation are notunderstood. Alveolar macrophages frompatients with sarcoidosis have an increasedability to function as antigen presenting cells(APC) and to support antigen driven prolifer-ation of autologous T lymphocytes, which isnot due to an upregulation ofHLA-DR mole-cules on the cell surface.14 Recently theexpression of adhesion molecules on alveolarmacrophages from patients with sarcoidosishas been reported.15 16 The present study wasundertaken to elucidate the contribution ofICAM-1 to the inflammatory process incorrelation with clinical and immunologicalparameters of disease activity in pulmonarysarcoidosis. Using a recently establishedELISA method which allows semiquantitativeanalysis of ICAM-1 levels on alveolarmacrophages as well as immunocytochemical

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staining, we determined ICAM-1 expressionin a group of untreated patients with pul-monary sarcoidosis and a group of healthyvolunteers.

MethodsSTUDY POPULATIONThe study population consisted of 17 consec-utive untreated patients with biopsy provenpulmonary sarcoidosis (radiological type 0,n = 1; type I, n = 9; type II, n = 7). Therewere 10 patients with active and seven withinactive disease. The assessment of clinicaldisease activity was performed according topreviously published criteria.8 Briefly, new orincreasing respiratory or systemic symptoms,a reproducible decline in vital capacity and/ordiffusing capacity of >10%, and new orprogressing infiltrates in the chest radiographwere accepted as signs of active disease (table1). Six healthy volunteers were studied ascontrols. There were no smokers in eithergroup which is notable as smoking influencesexpression of adhesion molecules.17 Ethicalcommittee approval and individual informedconsent were obtained. Clinical data andbronchoalveolar lavage cell counts aresummarised in table 2.

BRONCHOALVEOLAR LAVAGE AND CELLISOLATIONBronchoalveolar lavage was performed fromthe middle lobe using a flexible fibreopticbronchoscope under standard conditions.'8The lavage volume was 200 ml with a meanrecovery of 84%, which was similar inpatients and controls. The first 20 ml aliquotof the lavage fluid which is known to be con-taminated by bronchial secretions'9 was dis-carded and aliquots were pooled. A total cellcount was made by a haemocytometer

Table 1 Clinical characteristics ofpatients with activeand inactive sarcoidosis

Active Inactive

Chest radiographType 0

progressing 0 0stable 1 0

Type Iprogressing 6 0stable 1 2

Type IIprogressing 2 0stable 0 5

Lung function*Vital capacity

decreasing 5 0stable 5 6

Diffusing capacitydecreasing 7 0stable 3 6

Respiratory symptoms 7 1Systemic symptoms

Fever 5 0Erythema nodosum 4 0Arthritis/arthralgia 8 0

Extrathoracic sarcoidosisUveitis 1 0Skin involvement 1 0

*Data available only for 16 patients.

Table 2 Age, sex, and cell counts in bronchoalveolarlavage (BAL) fluid ofpatients and healthy volunteers

Patients Controls(n = 17) (n = 6)

Mean (SD) age (years) 38-2(15) 27(4)Sex (M:F) 6:11 6:0BAL fluid

Total cell count (x 106) 23(21) 11(3)Alveolar macrophages (%) 70(18) 90(7)Granulocytes (%) 1(1) 1(1)Eosinophils (%) 1(1)Lymphocytes (%) 28(19) 8(5)CD4+ (%) 81(7)CD4+ /CD8+ 8 8(4-6)

(Neubauer), then lavage cell differentialswere determined by Wright-Giemsa stains ofcytocentrifuge preparations (Cytospin II,Shandon). Cell viability was assessed usingtrypan blue dye exclusion. For differentiationof lymphocyte subsets (CD3, CD4, CD8)fluorescein conjugated monoclonal antibodies(Ortho Diagnostics, Beerse, Belgium) wereused. Lavage cells were washed twice withphosphate buffered saline and resuspendedat a concentration of 1 x 106 alveolar macro-phages/ml in M199 supplemented with fetalcalf serum (5%, Gibco), L-glutamine (2mmol/l, Gibco), and penicillin/streptomycin(1 mg/ml, Gibco).

ICAM-1 EXPRESSION ON ALVEOLARMACROPHAGES AND LYMPHOCYTESICAM-1 expression on alveolar macrophageswas determined by a sandwich ELISA tech-nique. Mononuclear lavage cells containingviable alveolar macrophages at a density of 1x 106/ml were seeded into 96-well flatbottom microtitre plates (Greiner, Niirtingen,Germany) for three hours (37°C, 5% carbondioxide), allowing the macrophages toadhere. Non-adherent cells were removed bygentle washing and serum-supplementedM199 was added. Incubation was continuedfor 16 hours at 37°C in a total humidifiedatmosphere containing 5% carbon dioxide.Cells were then fixed with 1% paraformalde-hyde (one hour, 21°C) and free binding siteswere blocked by adding a 2% solution ofbovine serum albumin (BSA) diluted inphosphate buffered saline (PBS, 0.1 moll,pH 7T2) for one hour (37°C). Themonoclonal mouse anti-ICAM-1 antibody(clone 84H10, Dianova, Hamburg,Germany, 1:1000 in 0-1% BSA/PBS) wasadded for two hours at 37°C. Peroxidaseconjugated goat antimouse antibody (Sigma,Deisenhofen, Germany, 1:1000 in 0-1%BSA/PBS) was used as developing antibody.Finally, ABTS (Sigma, 1 mg/ml) andhydrogen peroxide (2-8 ,ul, 30% H,O2/10 ml)in 0-1 molIl sodium acetate (pH 4-2) wasadded. The colour was allowed to developfor 120 minutes and plates were read witha microplate reader (Behring EL 311) 405nm.

Results were expressed as ELISA units(EU) from quadruplicate wells after subtrac-tion of non-specific background absorbancewhich was determined in the absence of the

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primary antibody. The number of adherentcells at the time of the ELISA was assessed inparallel wells by crystal violet staining. Usingthis method we found no differences betweenthe three groups of our study (data notshown).

In addition, ICAM-1 expression on bron-choalveolar cells was determined by immuno-cytochemical staining on cytocentrifugepreparations using the alkaline phosphatasemonoclonal mouse antialkaline phosphatase(APAAP) complex as described.20

SOLUBLE ICAM-1 (SICAM-1) SERUM LEVELSSerum was collected at the time of broncho-alveolar lavage and frozen at -20°C. sICAM-1 levels were determined with a commerciallyavailable kit (Serva, Heidelberg, Germany).

TNFa BIOASSAYTumour necrosis factor a (TNFa) activitywas measured with an L929 fibroblast lyticassay as previously described.2' Briefly, L929cells (6 x 104 well) were cultured in 96-wellflat bottom microtitre plates (Nunc) contain-ing serial dilutions of conditioned supernatantof sarcoid alveolar macrophages in the pres-ence of actinomycin D. After 20 hours ofincubation the remaining cells were stainedwith crystal violet. The TNFa concentration

Figure 1 ICAM-1expression of alveolarmacrophages from patients(n = 17) with active(n = 10) or inactive(n = 7) disease andhealthy volunteers (n = 6)as determined by ELISA.**p < 0 05 v control.

oc

0.0

a aa)

c-

c

Sarcoidosist-ZI-., ;,:,::5. ...........:~~-W b% t''?

Figure 2 Immunoalkaline phosphatase (APAAP) staining on cytospin preparations ofalveolar cellsfrom patient with sarcoidosis. Original magnification x 630, reduced to 82%dunng origination.

of the samples was quantified by comparingthe results with the linear portion of astandard curve obtained with human recom-binant TNFa (Serva, Heidelberg, Germany,specific activity 6-6 x 106 units/mg). Thespecificity of this bioassay was tested byneutralising peak samples with a goat anti-human TNFa antibody (obtained throughH Biermann, Bad Nauheim, Germany fromBritish Biotechnology).

SUPEROXIDE ANIONThe production of superoxide anion wasdetermined by a ferricytochrome c micro-assay.22 Briefly, 105 alveolar macrophageswere incubated in a 96-well microtitre platewith 100 ,umol ferricytochrome c ± 0-2 mg/mlsuperoxide dismutase, with or without 10mmol sodium fluoride or 100 nmol PMA. Allexperiments were done in triplicate. Opticaldensity was determined at 550 nm over aperiod of two hours at 30 minute intervals ona microplate reader.

STATISTICSNon-parametric statistics were used through-out the study. The Mann-Witney U test wasused for independent samples. Correlationswere made with the Spearman rank correla-tion, using the median as a marker of centraltendency. Differences were considered signifi-cant at p < 005.23

ResultsTOTAL AND DIFFERENTIAL CELL COUNTTable 2 shows the cell counts in broncho-alveolar lavage fluid. The total number ofcells recovered from the bronchoalveolarlavage fluid was increased in patients withpulmonary sarcoidosis. Furthermore, thepercentage of lymphocytes was significantlyincreased compared with the healthy controls(p < 0 005).

ICAM-1 ON ALVEOLAR CELLS AND SERUMLEVELS OF SICAM-1ICAM-1 expression could be detected onalveolar macrophages in all cases (fig 1).Significantly elevated expression was seen onalveolar macrophages from patients withsarcoidosis compared with controls (mean(SD) 0-74 (0.24) EU v 0-46 (0.12) EU, p <

0 05). Only patients with apparently activedisease showed significantly increased ICAM-1 levels on alveolar macrophages (0-82 (0.27)EU) compared with alveolar macrophagesfrom the controls.

Immunocytochemical staining confirmedthe results of the ELISA for the macrophagepopulation in the bronchoalveolar lavagefluid. Using the APAAP complex, ICAM-1expression could also be demonstrated onalveolar lymphocytes from patients withsarcoidosis (fig 2).

Serum levels of sICAM-1 ranged from 200to 700 ng/ml and were similar in patients andcontrols (490 (130) ng/ml v 320 (90) ng/ml).

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Table 3 Mean (range) data of macrophage activation in bronchoalveolar lavage fluid ofpatients with active andinactive sar>k,idosis and healthy controls

Sarcoidosis

Control Total Active Inactive

ICAM-1 (EU/105 AM) 0-46 (0-29-0 57) 0-78 (0 42-1 17) 0-82 (0-44-1-17) 0-64 (0 42-084)TNFa (units/1O6AM) 3 (0-10) 239 (5-1345) 319 (6-1345) 59 (5-141)0° (runol/I05 AM) 0-76 (0-1-40) 2-59 (0 37-479) 2-52 (1-04-79) 2-75 (0 37-3 97)

ICAM-1-intercellular adhesion molecule 1; EU-ELISA unit; TNFa tumour necrosis factor a; 02-superoxideanion; AM-alveolar macrophage.

No correlation between sICAM- 1 levels andICAM- 1 surface expression on alveolarmacrophages was demonstrated.

RELEASE OF TNFa AND OXIDANTS BY ALVEOLARMACROPHAGESAll alveolar macrophages from patients withsarcoidosis spontaneously released TNFa andfree oxygen radicals whereas only a smallamount of activity was detected in thesupematants of controls (table 3). TNFarelease of alveolar macrophages correlatedstrongly with the ICAM-1 surface expressionof those cells (r = 0-69, p < 0 05; fig 3). Thespontaneous release of free oxygen radicalswas significantly elevated in patients and didnot differ between subjects with active andinactive disease (table 3). No correlation withICAM- 1 expression could be shown.The intensity of the lymphocytic alveolitis

as assessed by absolute numbers and percent-age of CD4 + lymphocytes in broncho-alveolar lavage fluid was not related to theenhanced expression of ICAM-1 on alveolarmacrophages.

DiscussionPulmonary sarcoidosis is an inflammatorydisorder of unknown aetiology, characterisedby a mononuclear cell alveolitis and non-caseating granulomas, leading in some casesto interstitial fibrosis.'s'6 To elucidate thepossible role of adhesion molecules in thisdisease, we investigated the contribution ofICAM-1 to the process of recruitment andactivation of inflammatory cells in the lung.ICAM-1 levels on alveolar macrophages

were elevated in patients with pulmonarysarcoidosis. This increase was statisticallysignificant only in patients with active disease.

Figure 3 Correlationbetween the spontaneousrelease of TNFa andICAM-1 surfaceexpression of alveolarmacrophages insarcoidosis.

0) 10000

a0.

0

o

,, 1000

0

LL (z

r = 0-69, p < 0-05

mu~~~~~~~~

s~~~

*

)4 05 06 07 08 0.9 1 0 1-1 1 2

ICAM-1 (EU/105 alveolar macrophages)

No other statistical difference between thetwo groups of patients could be established,which is probably due to the limited numberof subjects in our study.

There are several possible mechanismsleading to the increased ICAM-1 levels wemeasured. One could be enhanced recruit-ment of blood monocytes into the pulmonarycompartment. In a recent study by Melis etal, however, no relation between ICAM- 1expression and different blood monocyteassociated markers on alveolar macrophageswas found."5 Furthermore, they found nodifference between ICAM-1 expression onblood monocytes from patients with sarcoido-sis and from healthy volunteers. This is inline with our findings that serum levels ofsICAM-1 were similar in our patients andcontrols.TNFa activity in the supematant of

alveolar macrophages correlated strongly withICAM-1 surface expression of those cells. Ithas been shown previously that TNFa andother cytokines induce ICAM- 1 on endo-thelial cells, whereas airway epithelial cells arepredominantly responsive to r-interferon.YWe recently showed that TNFa inducesICAM-1 on control alveolar macrophages invitro (unpublished observation). Thus, itseems likely that in sarcoidosis ICAM-1expression on alveolar macrophages isregulated in an autocrine manner, supportingthe concept that alveolar macrophages arepivotal cells in the pathogenesis andmaintenance of the inflammatory process inthe pulmonary compartment. Furthersupport for a local upregulation of ICAM-1was that serum levels of sICAM-1 were notrelated to the activation demonstrated onalveolar macrophages.Few studies exist which describe elevated

ICAM-1 levels on alveolar macrophages inpatients with sarcoidosis.15 16 However, to ourknowledge this is the first description ofincreased ICAM-1 expression in correlationwith other parameters of immunologicalactivity. We found no correlation betweenupregulated ICAM-1 expression of alveolarmacrophages to the number of CD4 +lymphocytes in bronchoalveolar lavage fluid,which is in agreement with previous work.'5Elevated ICAM-1 surface expression was notrestricted to alveolar macrophages in patientswith sarcoidosis, however, but could also bedemonstrated on alveolar lymphocytes.28 Thissuggests that the interaction between ICAM-1 and its ligands on macrophages and CD4 +lymphocytes functions reciprocally.

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Considering the strong correlation we

found between ICAM-1 expression andTNFa secretion of the activated macro-

phages, we conclude that upregulated ICAM-1 levels reflect disease activity in thepulmonary compartment. The fact that we

could not show a correlation with the numberof CD4 + lymphocytes agrees with otherstudies, suggesting different mechanisms ofactivation for the lymphocytic and macro-

phage component of the mononuclear cellalveolitis in pulmonary sarcoidosis.'5 24

It is not clear whether upregulated ICAM-1 expression on alveolar macrophages is an

early event during the development of themononuclear cell alveolitis or a result of theinappropriate cytokine secretion of alveolarmacrophages and lymphocytes in the pul-monary compartment. Further research isneeded to characterise the exact position ofICAM-1 in the inflammatory cascade insarcoidosis.

The authors acknowledge the expert technical assistance ofBirgitta Karlberg and Monika Losch.

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