Thorax 1991;46:835-838

Increased expression of growth factor genes formacrophages and fibroblasts in bronchoalveolarlavage cells of a patient with pulmonaryhistiocytosis X

J Barth, H Kreipe, H J Radzun, K Heidorn, W Petermann, B Bewig, M R Parwaresch

AbstractPulmonary histiocytosis X is the localmanifestation of a systemic disorder ofunknown cause characterised by infiltra-tion of Langerhans cell like histiocytesand parenchymal fibrosis. In a malesmoker with histologically proved his-tiocytosis X and functional impairmentbronchoalveolar lavage showed an in-crease in CD-l/OKT-6 antigen positivehistiocytes to 8%. Northern blot analysisof RNA from bronchoalveolar lavagecells showed an exaggerated expressionof the M-CSF gene and of the c-fms geneencoding for the corresponding receptor.An increased level of c-sis RNA, whichencodes the B chain of platelet derivedgrowth factor, was also found. Diffusereticulonodular infiltrates on the chestradiograph resolved with glucocorticoidtreatment and CD-1IOKT-6 antigenpositive histiocytes fell to 3%. Macro-phage colony stimulating factor, c-fmsand c-sis gene expression were reducedalmost to normal after treatment. Theresults suggest that macrophage colonystimulating factor and platelet derivedgrowth factor may have a role in theinitiation or maintenance of pathologicalreactions in pulmonary histiocytosis X.

Department ofMedicineJ BarthW PetermannB BewigDepartment ofPathology, Universityof Kiel, Kiel, GermanyH KreipeH J RadzumK HeidomM R ParwareschReprint requests to:Dr Jurgen Barth, IMedizinische Klinik,Schittenhelmstr 12, 2300Kiel, Germany.Accepted 18 July 1991

Pulmonary histiocytosis X is an idiopathicinterstitial lung disease characterised by infil-trating Langerhans like histiocytes and eosin-ophils." In most cases pulmonary infiltratesresolve with minimal or no residual fibrosis,though a few patients may progress to endstage pulmonary fibrosis. The underlyingimmunological mechanisms are not knownand why some cases progress to severe fibrosisis not clear. Lesions are characterised byproliferation of two major cellular con-

stituents, histiocytes and fibroblasts. Prolifera-tion and activation of both cell types are

regulated by soluble growth factors that havebeen identified and characterised.'8 131418 TheB chain of the fibroblast mitogen, plateletderived growth factor is encoded by the c-sisproto-oncogene.2 Bronchoalveolar lavage cellsof a patient with pulmonary histiocytosis Xwere analysed for the expression of the genefor this potent fibroblast growth factor and of

the genes for haematopoietic growth factors,which increase proliferation and activation ofmacrophages and macrophage related cells.813In addition, we analysed RNA expression ofinterleukin-3, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF) and of c-fms that encodes for the M-CSF receptor.

MethodsBronchoalveolar lavage was performed with200 ml sterile physiological saline. Forimmunophenotyping of lavage cells and tissuespecimens the monoclonal antibodies OKT-6and S-100 (Ortho, Heidelberg) were used.The immunostaining procedures were per-formed as described previously.

Northern blot analysisTotal cellular RNA was purified under ribo-nuclease inactivating conditions by the guan-idine thiocyanate-caesium chloride methodand analysed by electrophoresis of 10 ig RNAthrough 1-2% agarose formaldehyde gels foll-owed by Northern blot transfer to coatednylon membranes (Genescreen, New EnglandNuclear, Boston, Massachusetts." The clonedv-fms gene pSM3 was a gift from Dr C JSherr, Memphis, Tennessee.' A 1036 bpBgII/PstI fragment of the human M-CSFgene pcCSF-17 was obtained from Cetus Cor-poration, Emeryville, California.9 The clonedhuman GM-CSF gene and the gibboninterleukin-3 gene were obtained from theGenetics Institute, Cambridge, Massa-chusetts.'718 The integrity of the RNA sam-ples was checked by hybridisation with thecloned actin gene obtained as a gift from DrM W Kirschner, San Francisco.3DNA probes were labelled with 32P deoxy-

cytidine triphosphate (3000 Ci/mmol; Amer-sham, Braunschweig, Germany) by oligonu-cleotide priming (Multiprime, Amersham,Braunschweig) to specific radioactivities of1-5 x 109 -3 x 109 cpm/Lg DNA.6 Mem-branes were prehybridised at 42°C for 24hours in buffer consisting of 50% formamide,5 x sodium chloride sodium citrate (SSC),1% sodium dodecyl sulphate (SDS), 2 xDenhardt's solution, 25 mM phosphate buffer(pH 7 0), 5% dextran, and 200 Mg/ml salmon

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Figure 1 Chestradiographs (a) at thetime ofpresentation,displaying areticulonodular patternand (b) after threemonths' treatment withcorticosteroids, showing aconsiderable improvement.

sperm DNA. The RNA blots were hybridisedfor 24-48 hours at 42°C with 1 x 107 cpmlabelled DNA probe/ml hybridisation buffer(the same as prehybridisation buffer). Afterhybridisation the blots were washed twice in2 x SSC and 0-5% SDS for five minutes atroom temperature, twice in 0-2 x SSC and0.5% SDS for five minutes, and twice in0.1 x SSC and 0 5% SDS for 30 minutes at55'C. After drying blots were exposed to x rayfilms (Kodak XAR 5) with an intensifyingscreen at - 70°C for up to seven days.

Case reportA 43 year old man was admitted to hospitalwith' fever, cough, and chest pain of threemonths' duration. He smoked 30 cigarettes a

day but had not been exposed to any inhalation-hazards. Physical examination showed., noabnormalities. Investigations revealed a raisederythrocyte sedimentation rate and mildneutrophilia with 6% unsegmented-neutro-phils, 1% myelocytes, and 14% monocytes.Screening for various viruses including-humanimmunodeficiency virus, gave negative results.A chest radiograph showed bilateral.reticulo--nodular infiltrates (fig la) with a pneumothoraxat the apex of the right lung. Lung function

tests showed a decrease of vital capacity to 73%predicted. Single breath transfer factor forcarbon monoxide (TLCO) was 65% predicted.Cytological examination of bronchoalveolarlavage fluid showed 93% macrophages, mostladen with brown pigment, 6% lymphocytes,and 1% granulocytes. The proportion of CD-1/OKT-6 positive histiocytes was 8%.Examination of the transbronchial biopsyspecimens led to the diagnosis of an interstitiallung fibrosis of uncertain aetiology. Mor-phological changes typical of histiocytosis Xwere found in an open lung biopsy specimen (fig2). The disease appeared to be restricted to thelung as other investigations, including scin-tillation scanning ofthe bones, yielded negativeresults. Glucocorticoid treatment was startedwith prednisolone 80 mg/day, leading to rapiddisappearance of his symptoms. After threemonths the infiltrates in the chest radiographhad almost resolved except for slight residualreticulation (fig Ib). Vital capacity and TLCOhad improved considerably to values close tonormal. Cytological examination oflavage fluidat this time showed 94% alveolar macrophages,5% lymphocytes, and 1% granulocytes. Only3% of CD-1/OKT-6 positive cells could beseen. After a further three months repeatinvestigations gave almost identical results.

Northern blot analysis of growth factorgenesThese clinical findings were associated withchanges in the level of macrophage activationand profibrotic gene expression. The level of c-fms transcripts that code for the M-CSF recep-tor was high during the acute phase of thedisease, and fell after glucocorticoid treatment(fig 3). At this stage the amount of detectablefms-transcripts in alveolar macrophages weresimilar to that in normal alveolar macrophages.The exaggerated level of c-fms gene expressionwas accompanied by an enhanced auto-stimulatory expression of the M-CSF gene (fig3). RNA of other haemopoietic growth factorgenes, such as GM-CSF and Interleukin-3,were not detected in lavage fluid cells, eitherduring the acute phase or after resolution of theradiographic infiltrates. At the onset of thedisease the level of c-sis gene expression byhistiocytosis X lavage cells was high, and fellafter three months' of glucocorticoid treatment(fig 3).Thus lavaged cells obtained during the acute

phase of the disease showed enhanced expres-sion of the M-CSF gene, its correspondingreceptor gene, and the c-sis gene; all returnedto normal levels after resolution ofthe pulmon-ary infiltrates.

DiscussionCell to cell interaction in immunological res-ponses are mediated by soluble regulatoryfactors. Several such factors are produced bymonocytes and macrophages, which appear tohave a central role in the inflammation andtissue repair by controlling angiogenesis andfibroblast proliferation. An important factor is

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Increased expression ofgrowth factor genes for macrophages andfibroblasts in lavage cells with pulmonary histiocytosis X

Figure 2 (a) Open lungbiopsy specimen showingsevere interstitialinflammation with fibroticobliteration of the alveolarspaces. The inflammatoryinfiltrate consisted ofeosinophilic granulocytes,mononuclear cells, andmany histiocytes withindented, reniform nuclei,resembling Langerhanscells. (Haematoxylin andeosin.) (b) Immunohisto-chemically they display theS-100 antigen in theircytoplasma, confirmingthat they Were histiocytosisX cells.

(a)

The exact cellular source of c-sis remainsquestionable in this case and it is not clearwhether it could be ascribed to the CD-l/OKT-6-positive histiocytes or to stimulatedmacrophages. In addition to the c-sis expres-sion we analysed both the expression of the M-CSF gene and the gene for the correspondingM-CSF receptor encoded by the c-fms gene.As previously described in active pulmonarysarcoidosis," the acute phase of histiocytosis Xwas characterised by a higher level of c-fmsgene expression by lavage cells (fig 3), which isascribed to an increased influx of immaturemacrophage precursors into the alveoli.' 11

Normal alveolar macrophages display a lowlevel of c-fins expression,1" and activation ofmonocyte-macrophages is accompanied by areduced expression of the c-fms gene.'0

Contaminating lymphocytes in the lavagefluid are considered unlikely to have beenresponsible for the increased level of M-CSFgene transcripts as they made up only 6% of allcells.With the resolution of pulmonary infiltrates

caused by corticosteroid treatment M-CSFgene expression returned to normal levels (fig3).11 We conclude that autostimulation ofmonocyte/macrophages via M-CSF may rep-resent a central component of the abnormal

c-fms

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c-sis

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actin

(b) 2.0 0

platelet derived growth factor, a potentmitogen for fibroblasts,4, which is in partencoded by the c-sis proto-oncogene.2 Normalalveolar macrophages c-onstitutively express.low levels of the c-sis geiee.'2 Our results showthat in the proliferative phase of pulnonaryhistiocytosis X, when fibrotic-activity would beexpected, more transcripts of the c-sis genewere found in lavage fluid'cells (fig 3), suggest-ing a role for c-sis in-the induction of fibroticprocesses in this condition. This is supportedby reports of an exaggerated spontaneousrelease of platelet derived growth factor byalveolar macrophages in idiopathic pulmonaryfibrosis.'2

Figure 3 Northern blot analysis of lavagefluid cells,showing that the expression of the c-fms, c-sis, and M-CSF genes changes during the course of histiocytosis X.In the acute phase (left lane) high levels of the c-fms geneand the M-CSF gene were detected: the expression ofboth genes returned to normal" after resolution of thepulmonary infiltrates (right lane). The hybridisation withan actin gene showed that equal amounts of intact RNAhave been blotted. In addition, lavagefluid cells atpresentation had a higher level of expression of the c-sisproto-oncogene, which codesfor the B chain ofplateletderived growth*actor. Transcripts ofGM-CSF or theinterleukin-3 gene were notfound either in the acutephase of histiocytosis X or after resolution of theinfiltrates.

M-CSF

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GNI-CSF

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IL-3

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immunological reaction in pulmonary his-tiocytosis X as M-CSF is known to inducevarious relevant inflammatory mechanisms inmonocyte-macrophage populations.'6

This study was supported by Bundesministerium fur Forschungund Technologie grant 01 KE 8813.

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