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Clin. exp. Immunol. (1985) 60, 539-545. Human bronchoalveolar macrophage heterogeneity demonstrated by histochemistry, surface markers and phagocytosis V. A. GANT* & ANNE S. HAMBLINt *Chest Department and Department of Immunology, St Thomas's Hospital Medical School, London, UK (Acceptedfor publication 20 December 1984) SUMMARY Human alveolar macrophages (AM) were obtained by bronchoalveolar lavage from 18 patients with a variety of conditions. For each patient the percentages of AM showing the following properties were determined: (1) staining for the enzymes non-specific esterase (NSE) and acid phosphatase (ACP); (2) in vitro phagocytosis of Candida guillermondii; (3) expression of cell surface markers detected by two monoclonal antibodies (MoAb) (1 B5 and DA2) and two anti-monocyte/macrophage MoAb (UCHMI and RFD2); and (4) simultaneous phagocytosis of C. guillermondii and staining with the MoAb. In all patients the majority of AM were found to be Ta positive (90 + 10%) ACP positive (100%) and NSE positive (97 + 4%). In contrast a smaller proportion were UCHM 1 and RFD2 positive (77 + 11%, 68 + 12%) and less were phagocytic (37 + 177%). Whilst the total percentage of cells staining with the MoAb was unaltered by phagocytosis, the proportion of UCHM 1 or RFD2 positive cells was significantly higher in the phagocytic population than in the non-phagocytic population (90% and 85%, as opposed to 65% and 55%, P < 0 001). Thus only a proportion of Ta positive AM expressed monocyte/macrophage antigens and were phagocytic. Such heterogeneity may reflect different stages of macrophage maturation or the existence of macrophage subpopulations with functionally distinct roles in airways immunity. Keywords bronchoalveolar macrophages macrophage heterogeneity INTRODUCTION Bronchoalveolar lavage (BAL) provides a source of viable cells of monocyte/macrophage lineage derived from the airways of the lung in man (Finley et al., 1967). These cells appear to play important roles in mucosal immunity by being phagocytic (Cohen & Cline, 1971) and immunoregu- latory with both suppressive and stimulatory properties (Daniele et al., 1977; Yeager et al., 1982; Ettensohn & Roberts, 1983). Cells of the monocyte/macrophage lineage have been reported as showing heterogeneity with respect to histochemical and surface markers in various human tissues (Poulter, 1983; Poulter et al., 1983). Heterogeneity of lung macrophages has already been described in terms of size and the presence of smoking related inclusions (Mann et al., 1971) and phagocytosis (Reynolds, Kazmierowski & Newball, 1975; Hoidal, Schueling & Petersen, 1981). The aim of this study was to investigate AM heterogeneity in terms of the expression of immuno- and histochemical markers conventionally associated with cells of the monocyte/macrophage lineage (ACP and NSE Correspondence: Dr Anne S. Hamblin, Department of Immunology, St Thomas's Hospital Medical School, London SE1 7EH, UK. 539

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Page 1: Human bronchoalveolar macrophage heterogeneity demonstrated

Clin. exp. Immunol. (1985) 60, 539-545.

Human bronchoalveolar macrophage heterogeneitydemonstrated by histochemistry,surface markers and phagocytosis

V. A. GANT* & ANNE S. HAMBLINt *Chest Department and Department of Immunology,St Thomas's Hospital Medical School, London, UK

(Acceptedfor publication 20 December 1984)

SUMMARY

Human alveolar macrophages (AM) were obtained by bronchoalveolar lavage from 18patients with a variety ofconditions. For each patient the percentages ofAM showing thefollowing properties were determined: (1) staining for the enzymes non-specific esterase(NSE) and acid phosphatase (ACP); (2) in vitro phagocytosis of Candida guillermondii; (3)expression of cell surface markers detected by two monoclonal antibodies (MoAb) (1 B5and DA2) and two anti-monocyte/macrophage MoAb (UCHMI and RFD2); and (4)simultaneous phagocytosis of C. guillermondii and staining with the MoAb. In all patientsthe majority ofAM were found to be Ta positive (90 + 10%) ACP positive (100%) and NSEpositive (97+ 4%). In contrast a smaller proportion were UCHM1 and RFD2 positive(77 + 11%, 68 + 12%) and less were phagocytic (37 + 177%). Whilst the total percentage ofcells staining with the MoAb was unaltered by phagocytosis, the proportion ofUCHM 1or RFD2 positive cells was significantly higher in the phagocytic population than in thenon-phagocytic population (90% and 85%, as opposed to 65% and 55%, P < 0 001). Thusonly a proportion of Ta positive AM expressed monocyte/macrophage antigens and werephagocytic. Such heterogeneity may reflect different stages ofmacrophage maturation orthe existence of macrophage subpopulations with functionally distinct roles in airwaysimmunity.

Keywords bronchoalveolar macrophages macrophage heterogeneity

INTRODUCTION

Bronchoalveolar lavage (BAL) provides a source of viable cells of monocyte/macrophage lineagederived from the airways of the lung in man (Finley et al., 1967). These cells appear to playimportant roles in mucosal immunity by being phagocytic (Cohen & Cline, 1971) and immunoregu-latory with both suppressive and stimulatory properties (Daniele et al., 1977; Yeager et al., 1982;Ettensohn & Roberts, 1983). Cells of the monocyte/macrophage lineage have been reported asshowing heterogeneity with respect to histochemical and surface markers in various human tissues(Poulter, 1983; Poulter et al., 1983). Heterogeneity of lung macrophages has already been describedin terms of size and the presence of smoking related inclusions (Mann et al., 1971) and phagocytosis(Reynolds, Kazmierowski & Newball, 1975; Hoidal, Schueling & Petersen, 1981). The aim of thisstudy was to investigateAM heterogeneity in terms of the expression ofimmuno- and histochemicalmarkers conventionally associated with cells of the monocyte/macrophage lineage (ACP and NSE

Correspondence: Dr Anne S. Hamblin, Department ofImmunology, St Thomas's Hospital Medical School,London SE1 7EH, UK.

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enzymes, Ia antigens and monocyte/macrophage specific antigens) on the cells showing phagocyticfunction in vitro.

MATERIALS AND METHODS

Patients and BAL. Fibreoptic bronchoscopy was performed on 18 patients being investigatedfor a variety ofconditions shown in Table 1. Four 50 ml aliquots ofnormal saline buffered to pH 7-4with 8 4% sodium bicarbonate were instilled into a subsegmental bronchus ofthe middle lobe, rightlower lobe or lingula and gentle suction was applied after each aliquot. Samples were collected in acooled siliconized bottle and taken to the laboratory within 30 min. Differential cell counting,immunocytochemistry, and combined phagocytosis and immunocytochemistry was carried out onsamples from all patients. Immunocytochemistry using RFD2 was carried out on samples from 14patients and combined phagocytosis and immunocytochemistry using RFD2, on samples from 13patients.

Cells. The lavage fluid was centrifuged at 400g for 10 min. The cell pellet was washed once in coldserum free RPMI medium (GIBco) and resuspended in 5-10 ml RPMI. Total cell numbers wereassessed by crystal violet staining and viability was assessed by trypan blue dye exclusion. Afterdilution to 3 x 105 cells/ml, cytocentrifuge preparations (Shandon, GB) were made by spinning at200g for 10 minutes. The slides were air dried for at least 20 min and stained conventionally andhistochemically, or fixed in chloroform/acetone (50% vol./vol.) for 3 min, washed in phosphate-buf-fered saline (PBS, pH 7-2) for 10 min and stored at 40C in PBS containing 0-2% sodium azide forsubsequent immunocytochemical staining.

Conventional and histochemical staining. May-Grunwald-Giemsa (MGG) stain was used forassessment of lavage cell morphology. Previously described histochemical methods were used todemonstrate the presence of non-specific esterase, NSE (Yam et al., 1971) and acid phosphatase,ACP (Bancroft, 1982).

Monoclonal antibodies (MoAb) and immunocytochemical staining. The properties of the MoAbused are summarized in Table 2. Suitable titres of all antibodies were determined prior to the study,and single batches were used throughout. Fixed cytocentrifuged cells were incubated with 50 p1 ofantibody in a moist chamber at room temperature for 45 min, washed with PBS for 10 min,incubated with 50 y1 rabbit anti-mouse IgG (whole molecule) alkaline phosphatase conjugate(Sigma, cat. No. A 1902) for 45 min and washed again. The enzyme was allowed to develop aBrentamine Fast Red stain using naphthol AS.BI phosphoric acid substrate (Sigma, cat. No. 2250)applied for 45 min according to the method of Mason & Sammons (1978). Preparations were

Table 1. Patients studied and recovery ofAM

Diagnosis Number* % macrophages in lavaget

No diagnosist 8 (7) 934+4Carcinoma 5 (3) 83 6+997(biopsy proven)Rheumatoid lung 1 (0) 74Sarcoidosis 4 (0) 507 ±+23-8(biopsy proven)

* Number in parenthesis refers to number of smokers.t Expressed as a mean percentage+ s.d. of total

numbers of cells recovered in lavage.t Patients in this category were undergoing investiga-

tion for haemoptysis or abnormal chest X-ray; no diag-nosis was made and they remained well on follow-up.

V. A. Gant & Anne S. Hamblin540

Page 3: Human bronchoalveolar macrophage heterogeneity demonstrated

Human bronchoalveolar macrophage heterogeneityTable 2. MoAb used in this study

MoAb Specificity Reference

DA2 Anti-HLA DR antigen Brodsky, Parham & Bodmer (1980)(monomorphic)

lB5 Anti-Ia antigen Adams, Bodmer & Bodmer (1983)(o chain determinant)

RFD2 Stains> 90% peripheral Poulter & Duke (1983)blood monocytes, promonocytesand granulocytes andsome tissue macrophages

UCHM1 Stains > 85% peripheral Hogg et al. (1984)blood monocytes andsome tissue macrophages

54'

All MoAb were of IgGI subclass.

counterstained with Mayer's Haemalum and the slides were mounted in Farrant's medium.Control slides were incubated without the MoAb and with MoAb of irrelevant specificity. Thesame techniques applied to samples of peripheral blood mononuclear cells obtained byFicoll/Isopaque separation have shown agreement between the mean percentage of cells +s.d.identified as monocytes by MGG staining (22 +± %) NSE staining (21 + 8%) and binding theMoAb UCHM 1 (16+ 8%) and RFD2 (24+10%). Significant correlation coefficients were obtainedbetween the percentages of cells staining with UCHM I and MGG (r= 0436, P < 0005) andUCHM 1 and NSE (r= 0 617, P< 0005) in 59 samples but not with RFD2 and MGG or RFD2 andNSE (16 samples). Inspection of immunocytochemically stained preparations revealed RFD2positive mononuclear cells other than monocytes in some samples; however, most monocytes inpreparations stained with UCHM1 and RFD2.

Phagocytosis. A modification of the method of Pinching (1983) was used. One hundredmicrolitres of cell suspensions containing 1 x 105 macrophages were placed in LP3 tubes (LuckhamLtd.) with 60 ,l of C. guillermondii containing 12 x 105 organisms in RPMI and 100 p1 ofautologous serum. The tubes were gently rotated for 30 min at 37°C. The volume of the cellsuspension was adjusted to 500 yl with RPMI and cytocentrifuge preparations were made from 100p1 of this suspension. The slides were stained with MGG and NSE, or fixed and stainedimmunocytochemically. Control cells, to which C. guillermondii was not added, were processedsimilarly.

Assessment of results. All slides were counted by one operator using a x 100 oil objective. AMwere identified in all preparations by size (> 15 ,um) and morphology, i.e. irregularity of the cellmembrane and heterogeneous, often vacuolated cytoplasm. The percentage ofAM in any samplewas determined from a differential count of 500 cells in the MGG stained sides. In all otherpreparations 200 AM were counted. The number of NSE or ACP positive or immunocytochemi-cally stained AM was expressed as a percentage of the 200 AM examined.

In preparations which have been incubated with C. guillermondii in vitro, AM in which one ormore organism could be seen within a vacuole were considered to be phagocytic. In MGG and NSEstained preparations the number of such cells was expressed as a percentage of the total number ofAM. In preparations stained with MoAb both phagocytosis and staining were assessedindependently and the percentage of both phagocytic and non-phagocytic cells which had stainedwith each of the four MoAb was determined. Significant difference between means of differentgroups were assessed by Student's t-test.

RESULTSAppearance of conventionally and histochemically stainedAMViability exceeded 90% in most cases (range 70-100%). MGG staining revealed AM to be

Page 4: Human bronchoalveolar macrophage heterogeneity demonstrated

542 V. A. Gant & Anne S. Hamblin(a) (b)

100 _ _1000 II~~~~~~~~

80 + 5 80

60 -r P -60

40 - 40

T20 _ 20

I.Antibody IB5 DA2 UCHMI RFD2 Phagocytosis

(n= 18 ) (n =18 ) (n= 18) (n = 14)Specificity l lIl

Anti- H LA-DR Anti-monocyte/macrophage

Fig. 1. Percentage ofAM in individual patients which (a) stained with the MoAb indicated and (b) phagocytosedC. guillermondii. The solid bars represent the mean percentage for the group of patients.

heterogeneous in terms of size and appearance of the cell membrane and cytoplasm as has beenpreviously reported (Mann et al., 1971). As expected 97+4% of cells morphologically defined asAM stained for NSE and 100% ofAM stained for ACP. Furthermore heavy staining of the cells wasobtained after 4 min incubation for NSE and 30 min for ACP, contrasting with the 15 min and 1 h,respectively required to develop detectable staining in peripheral blood monocytes. No other cells(lymphocytes or polymorphs) stained for NSE or ACP. It was notable that not all AM recoveredfrom smokers contained phagocytosed tar particles; a proportion ofAM had clear cytoplasm andtherefore lacked evidence of in vivo phagocytic activity by light microscopy. These cells were ingeneral similar with a higher nuclear to cytoplasmic ratio. Taken together these results demonstratethat lung macrophages, which are all strongly ACP positive and NSE positive, show differingevidence of obvious in vivo phagocytosis.

Immunocytochemical staining with MoAbThe proportion ofAM from different patients staining with MoAb is shown in Fig. 1. The majorityofAM stained for lB5 (93 + 7%) and DA2 (87 + 1 1%); Ia antigens could therefore be detected onnearly all AM. There were no significant differences in expression of Ta in patients with differentdiseases nor with different smoking habits (1 B5; 93 + 6% in smokers, 95 + 8% in non-smokers andDA2; 85 + 9% in smokers, 91 + 8% in non-smokers). A smaller proportion of AM stained forUCHM 1 (77+ 1I I%) and RFD2 (68 + 12%) contrasting with the fact that these antigens areexpressed on nearly all peripheral blood monocytes (Table 2). Control slides not exposed to theMoAb did not stain, except for occasional endogenous alkaline phosphatase activity seen ineosinophils. No AM staining was seen in the control slides incubated with irrelevant MoAb.

PhagocytosisFig. I shows the individual phagocytosis results for the 18 patients. There was no consistentvariation in percentage with disease state in this small group of patients. A mean of 37% (± 17%) ofAM were phagocytic and no difference was found between percentages obtained from slides stainedwith MGG or NSE. Thus morphological definition of macrophages by MGG stained appeared tocorrelate with histochemical definition by the presence of NSE. AM showing in vitro phagocytosisusually contained products of phagocytosis acquired in vivo. Conversely, AM which did notobviously phagocytose in vitro often had clear cytoplasm. Viability, as measured by trypan blueexclusion, was unchanged after phagocytosis.

Page 5: Human bronchoalveolar macrophage heterogeneity demonstrated

Human bronchoalveolar macrophage heterogeneityOOIkP'0 02 0 01cP-O 02 P-O *001

100 -

80.

60 -

40 -

20

Antibody

Specificity

P<0.00I

IB5 DA2 UCHMI RFD2I-

Anti-la Anti-monocyte/mocrophl ~GP

Fig. 2. Mean percentage of phagocytic (ix) and non-phagocytic (0) AM which stained with the MoAb. Thevertical bars represent the standard deviation. The level of significance (P value) of differences in stainingbetween phagocytic and non-phagocytic populations for each antibody are indicated.

Table 3. Total percentages of cells staining with MoAb before and after phagocytosis

Before AfterMoAb phagocytosis phagocytosis Control*

UCHM1 77+11 80+10 81 +10RFD2 68 + 12 63 +14 66+ 10lB5 93+7 93+6 94+5DA2 89+11 87+10 85+12

* Cells processed as for phagocytosis but with-out C. guillermondii.

Combined phagocytosis and immunochemistryThe percentage of the phagocytic and non-phagocytic AM populations staining with MoAb isshown in Fig. 2. There was a significant difference (P < 0-001) in the percentage of phagocytic andnon-phagocytic cells which bound the anti-monocyte/macrophage MoAb (UCHM 1 and RFD2). Asmaller difference was obtained when these populations were stained for Ia antigens(0-01 < P < 002). A small number of unstained phagocytic cells was seen in most slides.

Table 3 shows that the total percentage of AM stained with MoAb before and afterphagocytosis was not significantly different, suggesting no selective loss of a population of cellsduring processing. Control slides incubated without C. guillermondii were similarly not significantlydifferent.

DISCUSSION

The majority of AM in agreement with other studies expressed Ta antigens (Mason et al., 1982;Moore et al., 1983) and were also strongly NSE positive and ACP positive (Poulter et al., 1983). Incontrast, the extent to which peripheral blood monocytes are Ia positive varies according todifferent authors between 30% and 100% (Albrechtson, 1981; Smith & Ault, 1981; Moore et al.,1983) and although most are NSE positive (Yam et al., 1971) and ACP positive (Poulter et al., 1983)

543

Page 6: Human bronchoalveolar macrophage heterogeneity demonstrated

V. A. Gant & Anne S. Hamblinstaining is weak by comparison with AM (Poulter et al., 1983, personal communication).Monocytes and macrophages found in other tissues also express these markers but to differingextents both in terms of numbers and in intensity. Such variations have been reported as beingassociated with both the tissues of origin and the extent of inflammation (Unanue, 1981; Poulter,1983). Ia positive, ACP positive macrophages have been described in tissue sections of skin involvedin delayed type hypersensitivity reactions and inflammatory bowel disease (Poulter, 1983). Theconsistently high percentage of ACP positive, Ia positive AM in this study, irrespective ofunderlying condition, suggests that this phenotype may not be unique to sites of inflammation andmay also be associated with cells involved in defense of exposed mucosal surfaces.

Such defence of mucosal surfaces is achieved in part by phagocytosis. In this study 37 + 17% ofthe AM phagocytosed C. guillermondii, contrasting with peripheral blood monocytes examined inthe same assay which were found to be greater than 90% phagocytic (data not shown). The methodused here differs from other phagocytic assays applied to AM in several respects: in particular, thetotal population of harvested cells was immediately examined. This contrasts with other workerswho have used adherent cells cultured in vitro prior to phagocytosis, and who have reported thatbetween 10% and 80% of the AM were phagocytic depending on the organism and serum conditionsused (Reynolds et al., 1975; Hoidal et al., 1981). It would appear that regardless of the assay, not allAM show phagocytosis in vitro. There are therefore la positive, ACP positive, NSE positivemacrophages in the lung which are not obviously phagocytic reflecting either their state of maturityor inability to perform this function.

A proportion of the AM stained with the anti-monocyte/macrophage MoAb UCHM I andRFD2 (Fig. 1), antibodies which stain many circulating blood monocytes (Hogg et al., 1984;Poulter & Duke, 1983). AM are thought to originate from bone marrow precursors (Thomas et al.,1976) and to be largely recruited from the peripheral blood monocyte pool (Blusse van Oud Alblas,van der Linden-Schrever & van Furth, 1983) although proliferation within the pulmonaryinterstitium has also been documented (Adamson & Bowden, 1980). Our observation that manyAM share antigens found on peripheral blood monocytes supports the view that they are derivedfrom the peripheral pool. It is unclear from this study whether AM exist which do not bind eitherUCHM1 or RFD2 since double labelling studies were not attempted. However, the existence ofUCHM 1 and RFD2 negative AM, suggests that these cells either originate from peripheral bloodmonocytes and subsequently lose their markers, or originate from altogether different precursors.

Simultaneous analysis of phagocytic ability and the expression ofUCHM 1 and RFD2 surfacemarkers demonstrated that a significantly greater proportion of phagocytic, as opposed tonon-phagocytic, cells expressed these markers (Fig. 2). A difference in staining was also seen usingthe anti-la antibodies although to a much less significant extent. It is unlikely that these findings canbe attributed to cell death since AM viability, as judged by trypan blue exclusion, did not declineafter the phagocytic assay, and the occasional unstained phagocytic AM was seen. Burmester et al.(1983) have described similar differences in staining patterns for both Ia and monocyte markers inthe phagocytic population from rheumatoid synovium. Ninety-one per cent of the latex ingestingcells were found to be positive for a MoAb known to stain 80% of peripheral blood monocytescontrasting with only I1% of the non-latex ingesting cells. Furthermore, some of the Ia positive,monocyte marker negative cells which were not phagocytic had morphological features of dendriticcells in culture. No attempts were made here to separate the cells or study them in vitro and the highlevel of lysosomal enzymes generally seen in AM is not consistent with the accepted morphologicalfeatures of dendritic cells. However, it is conceivable that some of the Ia positive non-phagocyticcells may represent a population of accessory cells with a similar active role in the generation ofimmune responses at the mucosal surface.

In conclusion, Ia positive, NSE positive, ACP positive AM may be divided into subpopulationsaccording to their expression of monocyte/macrophage surface markers and their ability tophagocytose. These subpopulations may be maturation and/or differentiation stages of cells ofsimilar lineage, or cells with different and distinctive origins; further analysis should reveal anyfunction they may have in the development and perpetuation of immune responses in the lung.

The skilled technical assistance of Ms Isobel Barbosa is gratefully acknowledged.

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Human bronchoalveolar macrophage heterogeneity 545

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