Int. J. Exp. Path. (I991) 72, 589-598

In-vitro interaction of human macrophages withPneumocystis carinii

Miguel Forte*t, Manjit Rahelut, Colin Stubberfieldt, Lesley Tomkins§,Alan Pithie*t and Dinakantha Kumararatnet

*East Birmingham Hospital, Birmingham; tDepartments of Immunology and §Physiology, Medical School,Birmingham and tWellcome Research Laboratories, Beckenham, UK

Received for publication 5 February I 99 IAccepted for publication I 9 June 1991

Summary. Pneumocystis carinii is an important opportunistic pathogen in patients withcompromised cell-mediated immunity. T-cell and macrophage function are believed to be ofprime importance in defence against this organism. The present ultrastructural study is aimedat the analysis of the interaction between human macrophages and P. carinii in vitro.Adherent peripheral blood mononuclear cells from healthy volunteers were exposed in vitro

to Pneumocystis derived from lungs of steroid-treated rats. The macrophages were harvested atdifferent intervals and studied by transmission and scanning electron microscopy.The material used for inoculation ofmacrophages was of identical morphology to previously

described P. carinii. When mixed with Pneumocystis in vitro, the macrophages appeared tomove towards the organism, extended pseudopods and ingested trophozoites and cysts. Within24 h, intracellular Pneumocystis underwent progressive degeneration inside macrophagevacuoles.

This study highlights the possible role of macrophages in host defence against P. carinii.

Keywords: Pneumocystis carinii, macrophages, electron microscopy

Pneumocystis carinii is a significant pathogenin patients with impaired cell-mediatedimmunity and gives rise to life threateninginterstitial pneumonia.

Originally described by Chagas (I909), P.carinii excited little interest until the firstcases of human disease caused by this organ-ism were recognized fifty years later byDeamer and Zollinger (I953). After that,several reports appeared in the literature andin I98I (Gottlieb et al. 198I) Pneumocystisacquired notoriety with its close association

with human immunodeficiency virus (HIV)infection.

Despite important advances in the diagno-sis (Hopewell I988) and therapy (Kovacs &Masur I988) of Pneumocystis pneumonia,very little is known about the biology, patho-genesis and mechanisms of protective immu-nity to this organism.

Immunosuppressive therapy (Hughes etal. I975) and HIV infection (Gottlieb et al.I 98 I), major risks for the development of P.carinii infection, are known to impair macro-

Correspondence: Dr Miguel Forte, Department of Communicable and Tropical Diseases, EastBirmingham Hospital, Bordesley Green East, Birmingham B9 5ST, UK.

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phage function (Masur et al. I982; Muller etal. 1990) which has previously been sug-gested as a major defence mechanismagainst Pneumocystis (Masur & Jones 1978;von Behren & Pesanti 1978).We have studied the interaction between

human macrophages and P. carinii usingelectron microscopy of peripheral blood de-rived macrophages infected with Pneumo-cystis in vitro.

Methods

Isolation of Pneumocystis carinii

Pneuunocystis organisms were obtained fromlungs of male Sprague-Dawley rats (Harlan-Olac, Bicester, Oxon., UK) which had beenimmunosuppressed by dexamethasone treat-ment (2 mg/l in the drinking water) for 8-Ioweeks. The animals were killed and the lungsperfused, via the pulmonary artery, withDulbeccos Modified Eagles Medium (Gibco)prior to homogenization for i min. Afterfiltering through a steel gauze, the homoge-nate was left to stand for 4 h at 40C in agravity sedimentation chamber containing a1-3% gradient of Histopaque (Sigma inHanks balanced salt solution supplementedwith 100 U/ml penicillin/streptomycin(Gibco)). Fractions were collected from thechamber and examined microscopically forPneumnocystis and rat cell content. The upperfractions were found on average to haveratios of around 3000: I parasite to hostcells. The preparation used in subsequentsteps contained trophozoites and cysts in aratio of I10: 1.

Separation of macrophages

Defibrinated venous blood was collectedfrom seven healthy adult volunteers from theDepartment of Immunology at the MedicalSchool, Birmingham, England. Peripheralblood mononuclear cells (PBMC) were separ-ated by density gradient separation on Ficoll/Hypaque (Pharmacia) (Boyum 1968). Thesewere washed three times in RPMI I 640

(Gibco) and resuspended in RPMI supple-mented with glutamine (2 mM), penicillin(I00 ,ug/ml), gentamicin (50 ,g/ml) andio% autologous serum (complete medium).

In-vitro infection of macrophages

Isolated PBMC, diluted to io7 cells/ml, wereincubated in 48-well tissue culture plates(Costar) in a humidified atmosphere of 5%CO2 in air at 3 70C for 24 h. The cells were leftto adhere to the bottom of the plastic wells orto glass cover slips placed on the bottom ofthe wells. All non-adherent cells wereremoved by washing and the media replacedwith I ml of fresh complete medium.Approximately i o% of the PBMC adhered asmacrophages. The macrophages were inocu-lated with a I07 suspension P. carinii. Thecells were then collected at regular intervalsup until 24 h. Macrophages were detachedfrom the bottom of the wells by scraping witha plastic policeman and centrifuged (700 gfor 5 min) into a pellet for transmissionelectron microscopy (TEM). For scanningelectron microscopy (SEM), the intact glasscover slip with the adherent cells was used insubsequent steps.

Electron microscopy

All samples were fixed in 2.5% gluteralde-hyde in o.o5 M phosphate buffer pH 7.3(osmolarity adjusted to 350 mosm withsucrose) for i h. Specimens for TEM werepost-fixed in i% osmium tetraoxide in o.o5%phosphate buffer for i h. Dehydration wascarried out in increasing concentrations ofacetone for SEM and alcohol for TEM. Mater-ial for SEM was critical-point dried in anEmscope CPD75o and sputter coated withapproximately 20 nm of platinum in anEmscope SCsoo. Material for TEM wasembedded in an Epon/Araldite resin mixture(Mollenhauer I964) and 70-Ioo-nm sec-tions were cut on a Reichert-Jung Ultracut E.The sections were collected on Formvar-coated slot grids and stained with 30%uranyl acetate in methanol for 7 min and

590

P. carinii and human macrophages

J~~~~~~~~~~14t

Fig. i. Pneumocystis carinii used for in-vitro inoculation ofmacrophages. A, Pneumocystis carinii cyst withinternal sporozoites (s) by transmission electron microscopy. Note the tubular structures seen in cross-section adjacent to cyst wall (uranyl acetate and Reynolds lead citrate, x 19 ooo). B, Cyst by scanningelectron microscopy. Tubular structures can be seen emerging from the cyst wall (platinum coated,x 1 5 ooo). C,D: Ruptured cysts, partially collapsed, with closely related trophozoites (t) (C, uranyl acetateand Reynolds lead citrate, x 14 ooo; D, platinum coated, x 20 000).

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Reynolds lead citrate for 7 min. All speci-mens were examined in a Jeol ioocx Tem-scan.

Kinetics of infection

To obtain an estimate number of intra andextra-cellular organisms, samples from twovolunteers were collected at i, 6, 12 and24 h. These experiments were performed inparallel with the same antigen preparation.Sections from those samples were examinedby TEM and randomly distributed fields werephotographed at a magnification of x 3ooo.A minimum of five negatives per sectionwere then scored for the numbers of P. cariniicysts and trophozoites inside and outsidecells. The number of cells with intracellularorganisms was also counted. The resultswere expressed per IOO cells.

Results

The material isolated from rat lung showedcysts and trophozoites of P. carinii. The cystsappeared round, 2-5 gm in diameter, withintracystic bodies o. 5-Ijim in diameter. Thecyst wall, measuring o. I jm on average, was

formed by three layers: an interior elec-tron-dense unit membrane, an intermediateelectron-lucent layer, and an exterior elec-tron-dense layer, as previously described(Campbell 19 72; Bedrossian I 989) (Fig. ia).Projections, apparently tubular in the SEM(Fig. ib), originated from the cyst wall andappeared in the sections of the TEM (Fig. ib)as circular structures, 0.05-0.2 jgm in dia-meter, with an outer electron-dense layerand an electron-lucent centre (Murphy et al.I977). Some of the cysts appeared to havereleased their content and acquired a cres-

cent shape, with trophozoites still closelyassociated (Fig. ic and d).The trophozoites, with identifiable nuclei

in the majority of cases, were polymorphicwith sizes between i and 4 jim. The cyto-plasm was limited by a single, thick unitmembrane which was markedly evaginated(Campbell 1972).

Control preparations of macrophagesshowed large cells with irregular shapednuclei I 5-20 gim total size.

P. carinii organisms isolated from lungs ofsteroid treated rats were presented in vitro toblood-derived human macrophages. Thirtyminutes after inoculation ofPneumocystis themacrophages appeared to move towardsPneumocystis with clump formation (Fig. 2a)and extension of pseudopods towards thecysts (Fig. 2b, c and d). The pseudopodsappeared to attach preferentially to tubularprojections rather than the intervening flatsurface of the cysts (Fig. 2d). Macrophagesalso phagocytosed trophozoites by evaginat-ing their cell membrane around the muchsmaller organism cell (Fig. 3). A clear exam-ple of this is seen in Fig. 3b where themacrophage membrane interdigitates withthe ruffled surface of the trophozoite.

This process resulted in most cells showinggenerally more than one vacuole containingcysts, trophozoites, tubular structures andamorphous material (Fig. 3a). Infected mac-rophages, with intracellular cysts at differentstages of degeneration, continued to attemptphagocytosis of extracellular organisms.

Figure 4 shows the number of organismsin the first 24 h after in-vitro infection for twodifferent volunteers. With individual A thereis an initial increase of extracellular organ-isms and a reduction in the numbers ofintracellular P. carinii. By I 2 h this pattern isreversed, with an increase in intracellularparasite count and a concomitant reductionin extracellular organisms. In the case ofindividual B, observations were made onlyup to 12 h. In this case, the numbers ofextracellular organisms fell progressivelywhile the intracellular uptake remainedsteady at about 30 organisms per IOO cells.In all circumstances, the trophozoites countreflected their initial higher number in rela-tion to cysts ( Io: i trophozoites to cysts). Theincrease in extracellular organisms seen inindividual A at I 2 h was due almost exclusi-vely to an increase in the trophozoites count.The percentage of cells with intracellular

organisms (Fig. 5) in individual A showed a

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P. carinii and human macrophages 593

_ _ _s_~~~~~~~~~~K.*~~~~~~~~~~~~~~~~~~~~~~~~f1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-1

Fig. 2. Initial stages of in-vitro infection of macrophages. A, clusters of human macrophages (in) withadherent cysts (c) (platinum coated, x iooo). B, macrophage with pseudopod extending towards a cyst(platinum coated, x 6ooo). C,D, Macrophages in the process of engulfing cysts (C, uranyl acetate andReynolds lead citrate, x 3600; D, platinum coated, x 5ooo).

M. Forte et al.594

Ar..Zr`.io-

v

P. carinii and human macrophages

cnE 400

CD

0) 300/\

0

u 200

E= 100 _z

0 1 6 12 24

Time (hours)

Fig. 4. Numbers of 0, 0, intracellular and *, *,extracellular organisms at different time pointsper ioo blood-derived macrophages from indi-viduals 0, 0, A and 0, * B.

variation in keeping with the numbers ofextracellular organisms. It started at an

initial high value of 36% and fell to a lowfigure of 8% at I 2 h, rising again after that to38%. In individual B the percentage of cellswith intracellular organisms remainedstable around I5% during the I 2 h of theexperiment.

Six to I 2 h after in-vitro infection of blood-derived macrophages with P. carinii, thenumber of cells showing phagocytosisincreased, resulting in several of them con-

taining one or more vacuoles with organ-

isms in progressive stages of degeneration(Fig. 3c). Extracellular cysts, isolated or inthe process of being engulfed by macro-

phages, could still be identified at 24 h (Fig.3d). Excess tubular structures whichappeared to be derived from protrusions of

-S32

E

24

0)

-c 16

0 1 6 12 24Time (hours)

Fig. 5. Percentage of monocyte-derived macro-

phages with intracellular organisms at differenttime points for individuals 0, A and *, B.

the membrane of Pneumocystis organisms,could be seen in the vicinity of the macro-

phages at all incubation times.

Discussion

P. carinii is a well recognized pathogen inimmunocompromised patients (Bruke &Good I973). In the overwhelming majorityof patients it causes pneumonia that, ifuntreated, is usually fatal. Recently, severalreports have also documented extra-pul-monary pneumocystosis (Telzak et al. I 990).The pathogenesis of P. carinii infection andthe mechanisms of host immunity to thisorganisms are not known at present. Impair-ment of T-cell (Shellito et al. I990) andmacrophage (Masur & Jones I978; von

Behren & Pesanti 19 78) function, seem to bemajor risk factors facilitating infection with

Fig. 3. Later stages of in-vitro infection of macrophages. A, Two hours after inoculation of Pneumocystiscarinii. Section ofhuman macrophage with vacuoles containing cysts (c) and collections of rod-like bodiesidentical to the tubular structures seen emerging from the organism (long arrow). The macrophage is inthe process of engulfing a trophozoite (short arrows) (uranyl acetate and Reynolds lead citrate, x 2900).B, detail from A showing intracellular tubular structures (long arrow) and the phagocytosis oftrophozoite (short arrow) (uranyl acetate and Reynolds lead citrate, x 6ooo). C, human macrophage,12 h after inoculation, with several intracellular cysts at different stages of degeneration (uranyl acetateand Reynolds lead citrate, x 3600). D, material collected 24 h after infection, showing a humanmacrophage with intracellular cysts in the process of ingesting another cyst. Extracellular cysts andtrophozoites are still identified (uranyl acetate and Reynolds lead citrate, X 2900).

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M. Forte et al.

this opportunist organism. The particularrelevance of the macrophage has been docu-mented in studies showing in-vitro phago-cytosis of Pneumocystis by macrophages frommice (Masur & Jones 1978) and rats (VonBehren & Pesanti 1978). Biopsy materialfrom human (Haselton et al. I98I) and rat(Yoneda & Walzer I980) lungs has alsoshown intracellular cysts of P. carinii withinpulmonary macrophages.At present there is no reliable long-term

in-vitro culture system for P. carinii; conse-quently, the best source of antigen remainsthe steroid-treated rat. Although aware ofantigenic differences between rat andhuman Pneumocystis there is a clear cross-reactivity between the antigens of the twoorganisms (Kovacs et al. I 988) to the point ofmonoclonal antibodies raised to rat Pneumo-cystis being used in the diagnosis of Pneumo-cystis pneumonia by immunofluorescence inbronchoalveolar lavage fluid from immuno-compromised patients (Gill et al. I987). Theantigen cross-reactivity and the lymphocyteproliferation (Forte et al. I 990) ofPBMC fromour volunteers to our antigen preparation(using a standard 3H-thymidine incorpora-tion assay (Kumararatne et al. I990)) justifythe use of rat Pneumocystis as a possiblemodel for host-organism interaction.The present study examines the interac-

tion of macrophages and P. carinii by infect-ing blood-derived human macrophages withrat Pneumocystis in vitro. The material usedfor inoculation of macrophage culture (Fig.i) had an identical morphology to previouslydescribed Pneumocystis organisms. In par-ticular, the tubular structures seen in associ-ation with the cyst wall and the trophozoitemembrane (Murphy et al. I977; Haselton etai. I98I), are apparently characteristic ofthis organism.The human monocyte-derived macro-

phages used in our study were obtained fromvenous blood of normal volunteers. Thesehad no prior activation other than culture asplastic-adherent cells for a period of 24 hbefore inoculation. It has recently beenshown that monocytes and monocyte-de-

rived macrophages, in the absence of lym-phocytes, produce tumour necrosis factoralpha (TNF-a) when exposed to P. carinii invitro to levels similar to those produced byEscherichia coli lipopolysaccharide (LPS)(Tamburrini et al. I 99 I). This effect could, inour system, contribute to the activation ofthe monocytes into macrophages.The organisms were added to human

monocyte-derived macrophages cultured inthe presence of I0% autologous serum.Subsequently, these cells extended pseudo-pods towards pneumocystis (Fig. 2) and by30 min, intracellular organisms could beidentified. The uptake of P. carinii seemed tolead to degeneration of cysts as several cellscould be identified with vacuoles containingcysts at different stages of degeneration (Fig.3c).The kinetics of P. carinii uptake as docu-

mented in Figs 4 and 5 reveal a differencebetween the two individuals studied.Although the effect of sampling and count-ing can not be completely excluded, thedifference may reveal a genuine variability inthe ability of macrophages to handle P.carinii infection. The cells from individual Ahave high levels of uptake initially, with littleapparent destruction of organisms. Indeed,the total numbers of organisms increased,due to an increase in trophozoites, raising thepossibility of content release from the cysts.The increase in total organism numbers andthe decrease in percentage of cells withintracellular organisms proceeds until 12 hwhen there is a reversal of these trends,possibly corresponding to further activationof the monocyte-derived macrophages thatare now becoming competent to controlinfection. In contrast, individual B shows apattern of steady uptake with reduction inthe number of extracellular organisms.Further studies are needed to clarify theapparent difference in behaviour of theblood-derived macrophage from the twoindividuals.

These results suggest that macrophagesfrom normal individuals may be capable ofdestroying P. carinii without activation by

596

P. carinii and human macrophages 597lymphocytes. The interpretation of degen-eration was on the basis of disintegration ofnormal architecture of the cysts seen byelectron microscopy, as described by otherauthors (von Behren & Pesanti 1978). Thatmacrophages kill P. carinii, however,remains presumptive at present in view ofthe absence of a reproducible method fordetermining the viability of Pneumocystis invitro. Also evident was the amount of intraand extra-cellular tubular structures asso-ciated with the surface of the organisms aspreviously reported (Yoneda & WalzerI980). The identity and function of suchstructures, which seemed to increase over a24-h period, in the presence ofmacrophages,is not known, although suggestions of theirrole in attachment and nutrition have beenmade previously (Murphy et al. I977; Bed-rossian I989).

After a 24-h culture period, intracellular,as well as extracellular, intact cysts could stillbe identified (Fig. 3d). The relatively shorttime period and the large number of organ-isms used in the inoculum could account forthis persistence. Alternatively, macrophagesmay need further activation by T-cell pro-ducts to be completely effective in destroyingP. carinii.

It seems likely that defence against P.carinii is multifactorial with the main con-tributors being the T-lymphocytes (Shellitoet al. I990), macrophages (Masur & Jones19 78; von Behren & Pesanti I 9 78) and theirproducts (Pesanti I990). Several factors mayinfluence the macrophage attachment anduptake of P. carinii-like antibodies (Masur &Jones 1978) and non-specific receptors, e.g.fibronectin (Pottratz & Martin I990). Theinfluence of several of these factors, the T-cellantigen specific responses, and cytokine pro-duction, are under investigation.

In conclusion, activated macrophages arerequired for the destruction of Pneumocystisin vivo. Impaired macrophage function dueto factors like steroid therapy or HIV infec-tion, may allow progression of P. cariniiinfection in the lungs and even result insystemic spread with transport of intracellu-

lar viable P. carinii in the blood. Theseobservations highlight the role of the macro-phage in host defence against Pneumocystiscarinii.

Acknowledgements

Dr Miguel Forte was supported by a grant bythe Calouste Gulbenkian Foundation, Lis-bon, Portugal. The authors wish to acknow-ledge Professor A.M. Geddes and Dr D. Cattyfor all their support.

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