Clin. exp. Immunol. (1989) 76, 41-46

The distribution of phenotypically distinct macrophage subsetsin the lungs of patients with cryptogenic fibrosing alveolitis

B. NOBLE, R. M. DU BOIS* & L. W. POULTER, Departments of Immunology and *Thoracic Medicine,Royal Free Hospital and School of Medicine, London

(Acceptedfor publication 2 December 1988)

SUMMARY

Monoclonal antibodies that identify phenotypically distinct macrophage subsets were used toanalyse the macrophages in lung biopsy specimens and bronchoalveolar lavage fluid from patientswith cryptogenic fibrosing alveolitis. Among the antibodies were RFD1, an interdigitating cellmarker, RFD7, a marker for mature tissue macrophages, and RFD9, which identifies epithelioid cellsas well as germinal centre macrophages. The lavage fluid was found to contain abnormally highnumbers ofcells staining with each ofthe antibodies, a finding that could be explained, at least in part,by an increased frequency of cells expressing more than one marker. In lung tissue macrophagephenotypes within the interstitium were found to differ significantly from those in the alveolar space.Most strikingly, cells bearing the antigen recognized by RFD9 were entirely absent from theinterstitial macrophage population, whereas the vast majority in the alveolar lumen were RFD9-positive. The discrete compartmentalization ofphenotypically different macrophages within the lungsuggests that macrophages may contribute differently to lung pathology in each microenvironment.The functional capacity of the unusual RFD9-positive alveolar macrophages remains to bedetermined, but their close association with the process of alveolar occlusion indicates a role in thefibrotic process.

Keywords macrophages lung disease bronchoalveolar lavage

INTRODUCTION

Using monoclonal antibodies to detect phenotypically distinctsubsets of immunocompetent cells, it has been found thatnormal bronchoalveolar lavage fluid contains heterogenouspopulations of macrophages and lymphocytes (Hance et al.,1985; Campbell, Poulter & du Bois, 1986). In interstitial lungdiseases, when inflammatory cells accumulate within the lung,the relative proportion of each mononuclear cell subset may

differ significantly from normal (Hance et al., 1985); Campbellet al., 1986; Paradis, Dauber & Rabin, 1986). The immunocom-petent cells recovered by bronchoalveolar lavage from patientswith sarcoidosis appear to provide a good sample of themononuclear cell subpopulations detectable in lung tissuebiopsies (Campbell, Poulter & du Bois, 1985a). Lung lavagepromises, therefore, to be a valuable clinical tool for monitoringinterstitial inflammation and evaluating the efficacy of therapyin sarcoidosis.

In cryptogenic fibrosing alveolitis the phenotypes of lym-

phocytes obtained by lung lavage also have been shown to

Correspondence: L. W. Poulter, Department of Immunology, RoyalFree Hospital School of Medicine, Pond Street, Hampstead, London,NW3 2QG, UK.

4'

mirror the lymphocyte subpopulations within lung tissue,although the correspondence may be imperfect in patients withsevere impairment of lung function (Paradis et al., 1986). Toassess bronchoalveolar lavage fluid from patients with cryptoge-nic fibrosing alveolitis as a source of lung macrophages, we havecompared the relative distribution of several different macro-phage subsets in lavage fluid and biopsy specimens. We wishedto know whether the macrophages in lavage fluid from patientswith cryptogenic fibrosing alveolitis were significantly differentfrom normal, and in particular, whether the presence of uniquesubsets might facilitate diagnosis of the condition. We wanted todetermine whether the macrophages recovered by lavage wererepresentative of those in the lung lesion, and, finally, wewondered whether the analysis of macrophage phenotypescould offer insights into the pathogenesis of the fibrotic lesion.

MATERIALS AND METHODS

Patients and normal controlsLung tissue samples were obtained from 11 patients on whomopen lung biopsies had been performed to confirm the diagnosisof cryptogenic fibrosing alveolitis. From five of those patients,cells obtained by bronchoalveolar lavage no earlier than 4 weeksprior to the biopsy were also available for study. In addition,

B. Noble, R. M. du Bois & L. W. Poulter

cells in lavage fluid were studied from three patients with biopsy-proven cryptogenic fibrosing alveolitis for whom lung tissuesamples were not available.

The patients (eight male and three female) ranged in age

from 51 to 70 years. In no case was there clinical or laboratoryevidence of a specific cause of alveolar fibrosis, althoughcoexistent connective tissue disease was documented in fourcases (systemic lupus erythematosus, 2; rheumatoid arthritis, 1;Sjogren's syndrome, 1). Biopsies and lung lavages were per-

formed as diagnostic procedures shortly after the initial presen-

tation with clinically significant lung disease. No patient was

receiving corticosteroids or other immunosuppressive drugs at

the time of investigation; all exhibited some degree of impair-ment of lung function; three were smokers. Most of the patientshave been described in detail in previous reports (Campbell et

al., 1985b, 1986). To obtain samples ofnormal lung macrophagepopulations for comparison, bronchoalveolar lavage was per-

formed on six healthy, non-smoking, medical student volunteers(five male, one female, mean age 26), who underwent bronchos-copy especially for this study. Informed consent was obtainedprior to all procedures, which were performed with the approvalof the Ethics Committee of the Royal Free Hospital.

Lung tissueBiopsy samples, taken from the right lower lobe, were dividedinto two pieces. One piece was fixed in formalin and processedfor routine histopathological study. For immunohistology theremainder was cut into several smaller pieces (0-5-1 -0 cm3) andfrozen with isopentane in a bath of liquid nitrogen. Sections 6gm thick were cut on a cryostat (Bright Instrument) at - 250C,air-dried for 1 h, fixed for 5 min in chloroform-acetone (1: 1),wrapped in plastic film and stored at - 20°C until used.

Bronchoalveolar lavage

One hour before the lavage, premedication consisting of 10 mgpapaveretum and 0-6 mg atropine was administered. The upper

respiratory tract was anaesthetized with topical 4% xylocaine. Afibreoptic bronchoscope (Olympus) was passed via the nose tothe lower respiratory tract which was anaesthetized locally. Theinstrument was wedged in a lateral segmental bronchus of theright lower lobe. A total of 180-240 ml of sterile 0-9% NaCl (pH7.4, 37°C) was introduced in 60 ml aliquots which were gentlyaspirated. The lung washings were collected into a sterilesiliconized bottle at 4°C.

Processing of lavage fluidThe total number of cells in an aliquot of untreated lavage fluidwas counted. Viability was always greater than 80%. Accordingto previously described procedures (Campbell et al., 1985a,1986), mucus strands were aspirated from the lavage fluid,which was then centrifuged at 450 g for 5 min to separate thecells. The supernatant fluid was removed and the cell pelletwashed twice with phosphate-buffered saline (PBS, pH 7.4). Thecells were resuspended at a concentration of 3 x 105/ml in PBS.Slides of 50-100 p1 aliquots of the cell suspensions were

prepared, with a cytocentrifuge (Shandon). After drying atroom temperature for 1 h, the slides were fixed in chloroform-acetone (1: 1) for 5 min, dried in air, wrapped in plastic film andstored at - 20°C until use. Some were stained with May-Grunwald-Giemsa preparation for differential cell counting.

Table 1. Monoclonal antibodies used in these studies

Antibody Major specificity indesignation normal lymphoid tissues Source

RFDl (p) Interdigitating cells and RFsubpopulation of B cells

RFD7 (y) Mature tissue macrophages RFRFD9 (y) Germinal centre macrophages RF

and granuloma macrophages(epithelioid cells)

UCHM 1 (y) Monocytes Dr N. HoggRFDR1 (y) HLA-DR RF

(Monocytes, macrophages,dendritic cells, B cells)

RF Royal Free Hospital School of Medicine.

ImmunocytologyThe monoclonal antibodies used in this study are shown inTable 1. Their pattern of reaction with cells in normal lymphoidtissues, summarized briefly in Table 1, has been describedextensively in earlier publications (Janossy et al., 1986; Poulteret al., 1986; Munro et al., 1987). With the exception of thehistocompatibility antigens identified by RFDR1, relativelylittle is known about the functional significance of the antigensrecognized by these monoclonal antibodies. RFD1 has beenshown to block mixed lymphocyte reactions (Poulter & Duke,1983). RFD7 appears to be associated with macrophagedifferentiation, emerging on peripheral blood monocytes onlyafter several days culture in vitro (Poulter et al., 1986). RFDI,RFD7, RFD9 and UCHM I identify distinctly different macro-phage subpopulations in normal lymphoid tissues. The ex-pression of more than one of those markers by individualnormal cells occurs infrequently, if it all.

Immunoperoxidase stainingThe procedure used in this laboratory has previously beendescribed in detail (Campbell et al., 1985a). Briefly, the slideswere incubated first for I h with each monoclonal antibodyappropriately diluted in PBS. After thorough washing in PBS,the slides were incubated for 45 min with peroxidase-conjugatedrabbit anti-mouse immunoglobulins (Dako). Exhaustive wash-ing to remove unbound antibodies was followed by treatmentfor 5-15 min with diaminobenzidine and hydrogen peroxidesolution. The slides were then counterstained with haematoxy-Ion for 30 seconds, dehydrated in graded alcohols and xylene,permanently mounted with coverslips and examined by lightmicroscopy (x 600). A minimum of 300 cells was examined oneach slide.

In lung biopsies macrophages were detected in two readilydistinguishable tissue compartments, the interstitium and thealveolar lumen. The relative proportion of each macrophagesubset in those locations was evaluated separately. For positivecontrols of the antibody specificity, sections of human tonsilwere stained. As a negative control, slides were incubated withnormal mouse serum and/or a monoclonal antibody to humanB lymphocytes, RFB4. Cells in both cytospins and lung biopsieswere counted without knowledge of the diagnosis. Each biopsyand cytospin was tested on at least two separate occasions with

42

Macrophages in fibrosing alveolitis

Table 2. Macrophages in bronchoalveolar lavage fluid*

Cryptogenic fibrosingStained with Normal alveolitis

RFD1 37±10 62+10RFD7 35+6 55+8RFD9 18+8 45+6UCHM1 8+2 2+3RFDR1 89+ 5 92+7RFDl andRFD7 10+5 17+6RFDl and RFD9 15+8 32+ 5

* Data expressed as percentage of total macro-phages.

Table 3. Macrophages in lung tissue in cryptogenicfibrosing alveolitis*

Tissue compartment

Stained with Alveolar space Interstitium

RFD1 75+20 40+15RFD7 43+15 38+12RFD9 85 + 18 < 1RFDR1 90+6 87+8UCHM1 < 1 8+2RFDl and RFD7 20+12 15+18RFD1 and RFD9 55+ 10 < 1

* Data expressed as percentage of total mac-rophages.

each of the monoclonal antibodies. In those repeated testsindividual mean values varied < 10%. The mean valuesreported here include all data collected in repeated tests.

Double immunofluorescence stainingProcedures for the simultaneous demonstration of two antigensby immunofluorescence microscopy have been previously pub-lished in detail (Janossy, Bofill & Poulter, 1985). Samples oflavage cells or lung tissue were incubated for 60 min with 50 pI ofa solution containing appropriate dilutions in PBS of twomonoclonal antibodies differing in heavy chain isotype. Forthese studies an IgM class RFD1 antibody was used incombination with IgG antibodies to RFD7 and RFD9, permit-ting the detection of cells bearing RFD1 as well as one of theother markers. After thorough washing in PBS, 50 p1 ofappropriately diluted rhodamine-labelled goat anti-mouse IgMand fluorescein-labelled goat-anti-mouse IgG (Southern Bio-technology Associates) were added together for 45 min. Theslides were washed once more with PBS, mounted with PBS/glycerol, and examined immediately with a fluorescence micro-scope (Zeiss). Positive and negative controls similar to thoseused for immunoperoxide staining were also used on immunof-luorescence tests. Two- to three-hundred cells were counted insuccessive high power fields, first with phase, and then fluores-

cence, optics, using appropriate barrier filters for rhodamineand fluorescein. The number of cells fluorescing either red orgreen or both was counted and expressed as a percentage of thetotal macrophage-like cells.

Statistical analysisAs all the data showed normal distributions around meanvalues, Student's t-test for non-paired data was applied todetermine statistical significance.

RESULTS

Bronchoalveolar lavageAs expected (Haslam et al., 1980; Hunninghake et al., 1981;Campbell et al., 1986), the differential count of cells frompatients with cryptogenic fibrosing alveolitis (12 +3% lympho-cytes, 69 + 8% macrophages, 19 + 7% neutrophils) revealed asignificant elevation (P<0-05), in neutrophils compared tohealthy controls (8+3% lymphocytes, 90+4% macrophages,2+ 1% neutrophils). The proportions of macrophages stainedwith RFD1, RFD7 and RFD9 (Table 2) were also abnormallyhigh (P < 0 05), resulting from the increased frequency of cellsbearing more than one marker. Increased expression of theantigen recognized by RFD9 was especially striking. In con-trast, there was no evidence of an influx of monocytes, asUCHM 1-positive cells remained relatively rare. HLA-DRexpression on lavage cells did not differ from normal. Althoughthe healthy controls in this particular study were younger thanthe patient population, the proportion of total macrophagesand those bearing antigens recognized by RFDI and RFD7were very similar to results obtained in previous investigationswith older and more heterogeneous controls (Campbell, et al.,1986).

Lung biopsiesAll the lung tissue specimens exhibited a 'mixed' pattern ofhistopathology: varying degrees of fibrosis were present inassociation with extensive mononuclear cell infiltration of thelung interstitium. In addition, clusters of mononuclear cells,most with a macrophage-like appearance, were observed withinthe alveolar lumen. In four biopsies the majority of alveoli werealmost entirely filled with mononuclear cells. In the remainder,the intraluminal accumulation of cells involved 10-30% ofalveoli. The macrophages in the lung interstitium (Table 3)consisted ofequal proportions of cells staining with RFD1 (Fig.la) and RFD7 (Fig. Ib), as well as a small fraction ofUCHM1-positive cells. The phenotypes of macrophages in the alveolarspace were quite different from the interstitium. Most of themacrophages within the alveolar lumen were positive for theRFD9 antigen. Expression ofRFD9 appeared to be restricted tomacrophages in the alveolar space (Fig. 2a). RFD9-positivecells were entirely absent from the interstitium, and, even whendeeply embedded in inflammatory or fibrotic lesions, RFD9-positive macrophages were always found to occupy the rem-nants ofan air space (Fig. 2a). Highly refractile connective tissuefibres, similar to those replacing the normal lung architecture,were detected between and around RFD9-positive macro-phages accumulated within the alveolar lumen (Fig. 2b). Thefraction of cells stained by RFD1 was also significantly elevated(P < 0-05) in the alveoli, where a high frequency was detected ofcells staining doubly with RFD1 and RFD9.

43

B. Noble, R. M. du Bois & L. W. Poulter

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Fig. 1. Light micrographs showing cells staining with RFD1 (a) and RFD7 (b) within cell clusters in the alveolar space (closed arrows)

and within the lung interstitium (open arrows) of a patient with cryptogenic fibrosing alveolitis (original magnification x 400).

Because the cell populations stained by RFDI, RFD7, andRFD9 (Tables 2 and 3) included cells stained by thoseantibodies alone, as well as cells expressing more than one

macrophage marker, a more detailed analysis was made of themacrophage subset composition in the tissue alveolar space andlavage fluid (Fig. 3). Data in Tables 2 and 3 were used to estimatethe proportion of cells doubly positive for RFD7 and RFD9,since that subset was not independently measured in these

experiments. By subtracting the fraction ofRFDl -positive cellsexpressing more than one marker from the total of RFDl-positive cells, the percentage expressing only RFD1 was deter-mined. The fraction ofcells expressing only RFD7 or RFD9 wassimilarly estimated. For that calculation the assumption wasmade that all cells stained with at least one of those monoclonalantibodies, as the contribution of monocytes (UCHMl-posi-tive) was negligible. Cells expressing RFDT or RFD7 alone,

44

Macrophages in fibrosing alveolitis

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Fig. 2. RFD9-positive cells (a) in clusters within the alveolar lumen(closed arrows). The remnant of a partially occluded airspace isindicated by closed arrowheads (original magnification x 400). Highlyrefractile fibres (b) (open arrows) were closely associated with RFD9-positive cells in the alveolar space and were also present in the fibroticinterstitium. The remnant of a partially occluded airspace is indicatedwith a closed arrowhead (original magnification x 400).

without coexpression of another marker, appeared to be absentfrom the alveolar space in tissue biopsies (Fig. 3). Cellsexpressing RFD9 in combination with RFD1 were more

frequent in the alveolar space than the lavage fluid.

DISCUSSION

The macrophages obtained by bronchoalveolar lavage frompatients with cryptogenic fibrosing alveolitis appeared to beboth quantitatively and qualitatively abnormal. Not only was

the frequency of cell staining with each of the subset markers,RFD 1, RFD7, and RFD9, significantly increased, but theproportions expressing two markers simultaneously were alsoelevated. Most striking was the increase in alveolar macro-

phages bearing the antigen recognized by RFD9. AlthoughRFD9 has proved to be a powerful probe for the identificationofepithelioid cells in granulomas (Munro, et al., 1987), it is not a

Fig. 3. A graphic representation of the relative proportions of differentmacrophage subsets present within the tissue alveolar space andrecovered by bronchoalveolar lavage fluid in cryptogenic fibrosingalveolitis. Cells doubly stained with RFD1 and RFD7 or RFD1 andRFD9 were counted directly (see Tables 2 and 3). The proportionexpressing antigens recognized by RFD1, RFD7, or RFD9 withoutcoexpression of other markers was calculated as described in the text(Results) as was the number of RRD7/RFD9-positive cells.

specific marker for granulomas, since it may also be expressedby specialized normal macrophages, including germinal centremacrophages and, as was shown here, a subset of normalalveolar macrophages. The fact that the RFD9 antigen is onlyexpressed on intraluminal macrophages in idiopathic pulmon-ary fibrosis, whereas it is a hallmark of interstitial pathology insarcoidosis (Munro, et al., 1987), suggests that local factorsinfluencing macrophage differentiation are quite different inthose two diseases of the lung.

Although highly abnormal, the macrophage subset compo-sition of lavage fluid was not sufficiently unique to be ofdiagnostic value, as similar populations have been observed insarcoidosis (Ainslie et al., 1988). No significant differences werenoted among patients with connective tissue disorders and thosefor whom pulmonary fibrosis was the only clinical problem,although such differences might be detected if larger numbers ofpatients and/or different macrophage markers were to bestudied. The functional capacity of the RFD9-positive cells inthe lavage fluid of patients with fibrosing alveolitis is presentlyunder investigation. In particular, it remains to be determinedwhether cells coexpressing RFD1 or RFD7 in combination withRFD9 antigens retain the characteristic antigen-presenting or

phagocytic capacity of their normal lymphoid tissue counter-parts. The abnormalities of alveolar macrophage function thathave been detected in fibrosing alveolitis (Hunninghake et al.,1981; Rennard et al., 1981; Bitterman et al., 1981; Wallaert et al.,1988) may prove to be restricted to cells ofsome phenotypes andnot others. Given the heterogeneity of lung macrophagesdescribed here, a rigorous evaluation of macrophage functionwill require study of the activity of individual subsets followingcareful separation.

Monoclonal antibodies have disclosed a discrete compart-mentalization of phenotypically discrete macrophage subpopu-lations within the lung in cryptogenic fibrosing alveolitis, a

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46 B. Noble, R. M. du Bois & L. W. Poulter

microanatomical distinction that was not detectable by conven-tional histology or histochemistry. Most macrophages locatedin interstitial sites belonged clearly either to the phenotypiccategory of interdigitating cells or mature tissue macrophages.In contrast, large numbers of cells in the alveolar lumen bearingthe phenotypic markers of interdigitating cells or mature tissuemacrophages also coexpressed the RFD9 subset marker. Theclose association of RFD9-positive cells with the process ofalveolar occlusion suggests that they are important in thedevelopment of the fibrotic lesion. That would be consistentwith the proposal of Basset et al. (1986) that intraluminalfibrosis is a relatively common finding in interstitial lungdisorders and, furthermore, that the intraluminal release ofgrowth factors by intraluminal macrophages may contributesignificantly to pathological processes involving the lung inter-stitium. The RFD9-positive macrophages that coexpress eitherRFD 1 or RFD7 antigens might be expected to have propertiesdistinguishing them from either classical antigen-presenting ormature tissue macrophages. Bronchoalveolar lavage fluid frompatients with cryptogenic fibrosing alveolitis could provide avaluable source of those cells for characterizing their functionalcapacity in vitro. It has been suggested that the microanatomicseparation of distinct subsets of phagocytic and dendritic cellswithin the normal spleen is indicative of functional specializa-tion (Buckley et al., 1986). Similarly, the physical separation ofmacrophage subpopulations within the lung opens the possibi-lity that the contribution of macrophages to lung pathology ineach microenvironment may be different. The detection ofmicroanatomical compartmentalization of lung macrophagesemphasizes the importance ofmeasuring the functional capacityof individual subsets for elucidating the pathological processesthat lead to fibrosis.

In the light of the microanatomical compartmentalization ofmacrophage phenotypes, the observation of significant discre-pancies between macrophage subsets in lavage fluid andinterstitial tissue was not surprising. Differences between mac-rophage subpopulations detected within the alveolar space intissue sections and those recovered by bronchoalevolar lavagemay have arisen from the failure of the lavage fluid to circulatefreely in alveoli packed with cells. That might also be themicroanatomical basis of the observation of Paradis, et al.(1986), that lymphocyte subsets in lavage fluid correlated poorlywith tissue lymphocytes in fibrosing alveolitis patients exhibit-ing reduced lung function.

ACKNOWLEDGMENTSDr Noble was supported by a Senior International Fogarty Fellowshipofthe N.I.H. The study was funded in part by a grant from the WellcomeTrust to L. W. Poulter.

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