Int.
J.
Exp. Path. (I991) 72, 589-598
In-vitro interaction of human macrophages with Pneumocystis 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 9 9 I Accepted for publication I 9 June 1 991
Summary. Pneumocystis carinii is an important opportunistic pathogen in patients with compromised cell-mediated immunity. T-cell and macrophage function are believed to be of prime importance in defence against this organism. The present ultrastructural study is aimed at 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 at different intervals and studied by transmission and scanning electron microscopy. The material used for inoculation of macrophages was of identical morphology to previously described P. carinii. When mixed with Pneumocystis in vitro, the macrophages appeared to move towards the organism, extended pseudopods and ingested trophozoites and cysts. Within 24 h, intracellular Pneumocystis underwent progressive degeneration inside macrophage vacuoles. 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 pathogen in patients with impaired cell-mediated immunity and gives rise to life threatening interstitial pneumonia. Originally described by Chagas (I909), P. carinii excited little interest until the first cases of human disease caused by this organism were recognized fifty years later by Deamer and Zollinger (I953). After that, several reports appeared in the literature and in I98I (Gottlieb et al. 198I) Pneumocystis acquired notoriety with its close association
with human immunodeficiency virus (HIV) infection. Despite important advances in the diagnosis (Hopewell I988) and therapy (Kovacs & Masur I988) of Pneumocystis pneumonia, very little is known about the biology, pathogenesis and mechanisms of protective immunity to this organism. Immunosuppressive therapy (Hughes et al. 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, East Birmingham Hospital, Bordesley Green East, Birmingham B9 5ST, UK.
589
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phage function (Masur et al. I982; Muller et al. 1990) which has previously been suggested as a major defence mechanism against Pneumocystis (Masur & Jones 1978; von Behren & Pesanti 1978). We have studied the interaction between human macrophages and P. carinii using electron microscopy of peripheral blood derived macrophages infected with Pneumocystis in vitro.
Methods Isolation of Pneumocystis carinii Pneuunocystis organisms were obtained from lungs of male Sprague-Dawley rats (HarlanOlac, Bicester, Oxon., UK) which had been immunosuppressed by dexamethasone treatment (2 mg/l in the drinking water) for 8-I o weeks. The animals were killed and the lungs perfused, via the pulmonary artery, with Dulbeccos Modified Eagles Medium (Gibco) prior to homogenization for i min. After filtering through a steel gauze, the homogenate was left to stand for 4 h at 40C in a gravity sedimentation chamber containing a 1-3% gradient of Histopaque (Sigma in Hanks balanced salt solution supplemented with 100 U/ml penicillin/streptomycin (Gibco)). Fractions were collected from the chamber and examined microscopically for Pneumnocystis and rat cell content. The upper fractions were found on average to have ratios of around 3000: I parasite to host cells. The preparation used in subsequent steps contained trophozoites and cysts in a ratio of I10: 1.
Separation of macrophages Defibrinated venous blood was collected from seven healthy adult volunteers from the Department of Immunology at the Medical School, Birmingham, England. Peripheral blood mononuclear cells (PBMC) were separated by density gradient separation on Ficoll/ Hypaque (Pharmacia) (Boyum 1968). These were washed three times in RPMI I 640
(Gibco) and resuspended in RPMI supplemented with glutamine (2 mM), penicillin (I00 ,ug/ml), gentamicin (50 ,g/ml) and io% autologous serum (complete medium). In-vitro infection of macrophages
Isolated PBMC, diluted to io7 cells/ml, were incubated 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 left to adhere to the bottom of the plastic wells or to glass cover slips placed on the bottom of the wells. All non-adherent cells were removed by washing and the media replaced with I ml of fresh complete medium. Approximately i o% of the PBMC adhered as macrophages. The macrophages were inoculated with a I07 suspension P. carinii. The cells were then collected at regular intervals up until 24 h. Macrophages were detached from the bottom of the wells by scraping with a plastic policeman and centrifuged (700 g for 5 min) into a pellet for transmission electron microscopy (TEM). For scanning electron microscopy (SEM), the intact glass cover slip with the adherent cells was used in subsequent steps. Electron microscopy All samples were fixed in 2.5% gluteraldehyde in o.o5 M phosphate buffer pH 7.3 (osmolarity adjusted to 350 mosm with sucrose) for i h. Specimens for TEM were post-fixed in i% osmium tetraoxide in o.o 5% phosphate buffer for i h. Dehydration was carried out in increasing concentrations of acetone for SEM and alcohol for TEM. Material for SEM was critical-point dried in an Emscope CPD75o and sputter coated with approximately 20 nm of platinum in an Emscope SCsoo. Material for TEM was embedded in an Epon/Araldite resin mixture (Mollenhauer I964) and 70-Ioo-nm sections were cut on a Reichert-Jung Ultracut E. The sections were collected on Formvarcoated slot grids and stained with 30% uranyl acetate in methanol for 7 min and
P. carinii and human macrophages
59I
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4t
Fig. i. Pneumocystis carinii used for in-vitro inoculation of macrophages. A, Pneumocystis carinii cyst with internal sporozoites (s) by transmission electron microscopy. Note the tubular structures seen in crosssection adjacent to cyst wall (uranyl acetate and Reynolds lead citrate, x 19 ooo). B, Cyst by scanning electron 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 acetate and Reynolds lead citrate, x 14 ooo; D, platinum coated, x 20 000).
M. Forte et al. 592 Control preparations of macrophages Reynolds lead citrate for 7 min. All speciTem- showed large cells with irregular shaped mens were examined in a Jeol nuclei I 5-20 gim total size. scan. P. carinii organisms isolated from lungs of steroid treated rats were presented in vitro to Kinetics of infection blood-derived human macrophages. Thirty To obtain an estimate number of intra and minutes after inoculation of Pneumocystis the extra-cellular organisms, samples from two macrophages appeared to move towards volunteers were collected at 6, 12 and Pneumocystis with clump formation (Fig. 2a) 24 h. These experiments were performed in and extension of pseudopods towards the parallel with the same antigen preparation. cysts (Fig. 2b, c and d). The pseudopods Sections from those samples were examined appeared to attach preferentially to tubular by TEM and randomly distributed fields were projections rather than the intervening flat photographed at a magnification of x 3ooo. surface of the cysts (Fig. 2d). Macrophages A minimum of five negatives per section also phagocytosed trophozoites by evaginatwere then scored for the numbers of P. carinii ing their cell membrane around the much cysts and trophozoites inside and outside smaller organism cell (Fig. 3). A clear examcells. The number of cells with intracellular ple of this is seen in Fig. 3b where the organisms was also counted. The results macrophage membrane interdigitates with were expressed per IOO cells. the ruffled surface of the trophozoite. This process resulted in most cells showing more than one vacuole containing generally Results cysts, trophozoites, tubular structures and The material isolated from rat lung showed amorphous material (Fig. 3a). Infected maccysts and trophozoites of P. carinii. The cysts rophages, with intracellular cysts at different appeared round, 2-5 gm in diameter, with stages of degeneration, continued to attempt intracystic bodies o. 5-I jim in diameter. The phagocytosis of extracellular organisms. cyst wall, measuring o. I jm on average, was Figure 4 shows the number of organisms formed by three layers: an interior elec- in the first 24 h after in-vitro infection for two tron-dense unit membrane, an intermediate different volunteers. With individual A there electron-lucent layer, and an exterior elec- is an initial increase of extracellular organtron-dense layer, as previously described isms and a reduction in the numbers of (Campbell 9 72; Bedrossian I 989) (Fig. ia). intracellular P. carinii. By I 2 h this pattern is Projections, apparently tubular in the SEM reversed, with an increase in intracellular (Fig. ib), originated from the cyst wall and parasite count and a concomitant reduction appeared in the sections of the TEM (Fig. ib) in extracellular organisms. In the case of as circular structures, 0.05-0.2 jgm in diaindividual B, observations were made only meter, with an outer electron-dense layer up to 12 h. In this case, the numbers of and an electron-lucent centre (Murphy et al. extracellular organisms fell progressively I977). Some of the cysts appeared to have while the intracellular uptake remained released their content and acquired a cressteady at about 30 organisms per IOO cells. cent shape, with trophozoites still closely In all circumstances, the trophozoites count associated (Fig. and d). reflected their initial higher number in relaThe trophozoites, with identifiable nuclei tion to cysts ( Io: i trophozoites to cysts). The in the majority of cases, were polymorphic increase in extracellular organisms seen in with sizes between and 4 jim. The cytoindividual A at I 2 h was due almost exclusiplasm was limited by a single, thick unit vely to an increase in the trophozoites count. membrane which was markedly evaginated The percentage of cells with intracellular (Campbell 1972). organisms (Fig. 5) in individual A showed a ioocx
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2. Initial stages of in-vitro infection of macrophages. A, clusters of human macrophages (in) with adherent cysts (c) (platinum coated, x i ooo). B, macrophage with pseudopod extending towards a cyst (platinum coated, x 6ooo). C,D, Macrophages in the process of engulfing cysts (C, uranyl acetate and Reynolds lead citrate, x 3600; D, platinum coated, x 5ooo).
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variation in keeping with the numbers of extracellular organisms. It started at an initial high value of 36% and fell to a low figure of 8% at I 2 h, rising again after that to 38%. In individual B the percentage of cells with intracellular organisms remained stable around I5% during the I 2 h of the experiment. Six to I 2 h after in-vitro infection of bloodderived macrophages with P. carinii, the number of cells showing phagocytosis increased, resulting in several of them containing one or more vacuoles with organisms in progressive stages of degeneration (Fig. 3c). Extracellular cysts, isolated or in the process of being engulfed by macrophages, could still be identified at 24 h (Fig. 3d). Excess tubular structures which appeared to be derived from protrusions of
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Fig. 5. Percentage of monocyte-derived macrophages with intracellular organisms at different time points for individuals 0, A and *, B.
the membrane of Pneumocystis organisms, could be seen in the vicinity of the macrophages at all incubation times. Discussion a well recognized pathogen in immunocompromised patients (Bruke & Good I973). In the overwhelming majority of patients it causes pneumonia that, if untreated, is usually fatal. Recently, several reports have also documented extra-pulmonary pneumocystosis (Telzak et al. I 990). The pathogenesis of P. carinii infection and the mechanisms of host immunity to this organisms are not known at present. Impairment of T-cell (Shellito et al. I990) and macrophage (Masur & Jones I978; von Behren & Pesanti 9 78) function, seem to be major risk factors facilitating infection with
P. carinii is
1
Fig. 3. Later stages of in-vitro infection of macrophages. A, Two hours after inoculation of Pneumocystis carinii. Section of human macrophage with vacuoles containing cysts (c) and collections of rod-like bodies identical to the tubular structures seen emerging from the organism (long arrow). The macrophage is in the 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 of trophozoite (short arrow) (uranyl acetate and Reynolds lead citrate, x 6ooo). C, human macrophage, 1 2 h after inoculation, with several intracellular cysts at different stages of degeneration (uranyl acetate and Reynolds lead citrate, x 3600). D, material collected 24 h after infection, showing a human macrophage with intracellular cysts in the process of ingesting another cyst. Extracellular cysts and trophozoites are still identified (uranyl acetate and Reynolds lead citrate, X 2900).
596
M. Forte et al. this opportunist organism. The particular rived macrophages, in the absence of lymrelevance of the macrophage has been docuphocytes, produce tumour necrosis factor mented in studies showing in-vitro phagoalpha (TNF-a) when exposed to P. carinii in cytosis of Pneumocystis by macrophages from vitro to levels similar to those produced by mice (Masur & Jones 1978) and rats (Von Escherichia coli lipopolysaccharide (LPS) Behren & Pesanti 1978). Biopsy material (Tamburrini et al. I 99 I). This effect could, in from human (Haselton et al. I98I) and rat our system, contribute to the activation of (Yoneda & Walzer I980) lungs has also the monocytes into macrophages. shown intracellular cysts of P. carinii within The organisms were added to human pulmonary macrophages. monocyte-derived macrophages cultured in At present there is no reliable long-term the presence of I0% autologous serum. in-vitro culture system for P. carinii; conse- Subsequently, these cells extended pseudoquently, the best source of antigen remains pods towards pneumocystis (Fig. 2) and by the steroid-treated rat. Although aware of 30 min, intracellular organisms could be antigenic differences between rat and identified. The uptake of P. carinii seemed to human Pneumocystis there is a clear crosslead to degeneration of cysts as several cells reactivity between the antigens of the two could be identified with vacuoles containing organisms (Kovacs et al. I 988) to the point of cysts at different stages of degeneration (Fig. monoclonal antibodies raised to rat Pneumo3c). cystis being used in the diagnosis of PneumoThe kinetics of P. carinii uptake as docucystis pneumonia by immunofluorescence in mented in Figs 4 and 5 reveal a difference bronchoalveolar lavage fluid from immuno- between the two individuals studied. compromised patients (Gill et al. I987). The Although the effect of sampling and countantigen cross-reactivity and the lymphocyte ing can not be completely excluded, the proliferation (Forte et al. I 990) of PBMC from difference may reveal a genuine variability in our volunteers to our antigen preparation the ability of macrophages to handle P. (using a standard 3H-thymidine incorpora- carinii infection. The cells from individual A tion assay (Kumararatne et al. I990)) justify have high levels of uptake initially, with little the use of rat Pneumocystis as a possible apparent destruction of organisms. Indeed, model for host-organism interaction. the total numbers of organisms increased, The present study examines the interac- due to an increase in trophozoites, raising the tion of macrophages and P. carinii by infectpossibility of content release from the cysts. ing blood-derived human macrophages with The increase in total organism numbers and rat Pneumocystis in vitro. The material used the decrease in percentage of cells with for inoculation of macrophage culture (Fig. intracellular organisms proceeds until 12 h i) had an identical morphology to previously when there is a reversal of these trends, described Pneumocystis organisms. In par- possibly corresponding to further activation ticular, the tubular structures seen in associ- of the monocyte-derived macrophages that ation with the cyst wall and the trophozoite are now becoming competent to control membrane (Murphy et al. I977; Haselton et infection. In contrast, individual B shows a ai. I98I), are apparently characteristic of pattern of steady uptake with reduction in this organism. the number of extracellular organisms. The human monocyte-derived macro- Further studies are needed to clarify the phages used in our study were obtained from apparent difference in behaviour of the venous blood of normal volunteers. These blood-derived macrophage from the two had no prior activation other than culture as individuals. plastic-adherent cells for a period of 24 h These results suggest that macrophages before inoculation. It has recently been from normal individuals may be capable of shown that monocytes and monocyte-dedestroying P. carinii without activation by
P. carinii and human macrophages lymphocytes. The interpretation of degeneration was on the basis of disintegration of normal architecture of the cysts seen by electron microscopy, as described by other authors (von Behren & Pesanti 1978). That macrophages kill P. carinii, however, remains presumptive at present in view of the absence of a reproducible method for determining the viability of Pneumocystis in vitro. Also evident was the amount of intra and extra-cellular tubular structures associated with the surface of the organisms as previously reported (Yoneda & Walzer I980). The identity and function of such structures, which seemed to increase over a 24-h period, in the presence of macrophages, is not known, although suggestions of their role in attachment and nutrition have been made previously (Murphy et al. I977; Bedrossian I989). After a 24-h culture period, intracellular, as well as extracellular, intact cysts could still be identified (Fig. 3d). The relatively short time period and the large number of organisms used in the inoculum could account for this persistence. Alternatively, macrophages may need further activation by T-cell products to be completely effective in destroying P. carinii. It seems likely that defence against P. carinii is multifactorial with the main contributors being the T-lymphocytes (Shellito et al. I990), macrophages (Masur & Jones 1 9 78; von Behren & Pesanti I 9 78) and their products (Pesanti I990). Several factors may influence the macrophage attachment and uptake of P. carinii-like antibodies (Masur & Jones 1978) and non-specific receptors, e.g. fibronectin (Pottratz & Martin I990). The influence of several of these factors, the T-cell antigen specific responses, and cytokine production, are under investigation. In conclusion, activated macrophages are required for the destruction of Pneumocystis in vivo. Impaired macrophage function due to factors like steroid therapy or HIV infection, may allow progression of P. carinii infection in the lungs and even result in systemic spread with transport of intracellu-
597
lar viable P. carinii in the blood. These observations highlight the role of the macrophage in host defence against Pneumocystis carinii.
Acknowledgements Dr Miguel Forte was supported by a grant by the Calouste Gulbenkian Foundation, Lisbon, Portugal. The authors wish to acknowledge Professor A.M. Geddes and Dr D. Catty for all their support. References BEDROSSIAN C.W.M. (I989) Ultrastructure of Pneumocystis carinii: a review of internal and surface characteristics. Semin. Diagn. Pathol. 6, 212-237. BOYUM A. (I968) Ficoll Hypaque method for separating mononuclear cells from human blood. Clin. Lab. Invest. 21 (SuPPI. 97), 77-89. BRUKE B.A. & GOOD R.A. (I973) Pneumocystis carinii infection. Medicine 52, 23-51. CAMPBELL W.G. (I 972) Ultrastructure of Pneumocystis in human lung: life cycle of human pneumocystosis. Arch. Pathol. 93, 312-324. CHAGAS C. (i 909) Nova tripanozomiaze humana. Mem. Inst. Oswaldo Cruz I, I59-2I8. DEAMER W.C. & ZOLLINGER H.V. (I 9 5 3) Interstitial 'Plasma Cell' pneumonia of premature and young infants. Pediatrics 12, II-22. FORTE M., PITHIE A., RAHELIJ M., STUBBERFIELD C., KUMARARATNE D. & GEDDES A. (I990) Cytolytic T-cells to Pneumocystis carinii infected macrophages. VI International AIDS Conference, Abstract SA 243. GILL V.J., EVANS G., STOCK F., PARRILO J.E., MASUR H. & KoVAKS J.A. (I987) Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J. Clin. Microbiol. 25, I837-I840. GOTTLIEB M.S., SCHROFF R. & SCHANKER H.M. (i 98I) Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men. N. Engi. 1. Med. 305, 14251431. HASELTON P.S., CURRY A. & RANKIN E.H. (I.98I) Pneumocystis carinii pneumonia: a light microscopal and ultrastructural study. J. Clin. Pathol. 34, II38-I 146. HOPEWELL P.C. (I988) Pneumocystis carinii pneumonia: diagnosis. J. Infect. Dis. 157, II15III9.
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HUGHES W.T., FELDMAN S. & AUR R.J.A. (I 975)
Intensity of immunosuppressive therapy and the incidence of Pneumocystis carinii pneumonitis. Cancer 36, 2004-2009. KOVACS J.A., HALPERN J.L. & SWAN J.C. (I988) Identification of antigens and antibodies specific for Pneumocystis carinii. 1. Imnmunol. 140, 2023-2031. KOVACS J.A. & MASUR H. (1988) Pneumocystis carinii pneumonia: therapy and prophylaxis. J. Infect. Dis. 158, 254-259. KIJMARARATNE D.S., PITHIE A.S., DRYSDALE P., GASTON J.S.H., KIESSLING R., ILES P.B., ELLIS C.J., INNES J. & WISE R. (I990) Specific lysis of mycobacterial antigen-bearing macrophages by Class II. MHC-restricted polyclonal T-cell lines in healthy donors or patients with tuberculosis. Clin. Exp. Med. 80, 314-323. MASIJR H. & JONES T.C. (1978) The interaction in vitro of Pneumocystis carinii with macrophages and L-cells. J. Exp. Med. 147, 1 57-I 70. MASIJR H., MURRAY H.W. & JONES T.C. (I982) Effect of hydrocortisone on macrophage response to lymphokine. Infect. Immun. 35, 709714. MOLLENHAUER H.H. (I 964) Plastic embedding mixtures for use in electron microscopy. Stain. Teclinol. 39, 11 '-I I4. MIJLLER F., ROLLAG H., GAUDERNACK G. & FROLANOS S.S. (I990) Impaired in-vitro survival of monocytes from patients with HIV infection. Clin. Exp. Immunol. 8I, 25-30.
MURPHY M.J., PIFER L.L. & HUGHES W.T. (I 9 7 7) Pneumocystis carinii in vitro: a study by scanning electron microscopy. Am. 1. Pathol. 86,
387-402.
PESANTI E.L. (I990) Interaction of cytokines and alveolar cells with Pneumocystis carinii in vitro. J. Infect. Dis. I63, 6II-6i6. POTTRATZ S.T. & MARTIN II W.J. (1990) Mechanism of Pneumocystis carinii attachment to cultured rat alveolar macrophages. J. Clin. Invest. 86, I678-I683. SHELLTO J., SIJZARA V.V. & BLUMENFELD W. (I 990) A new model of Pneumnocystis carinii infection in mice selectively depleted of helper T-lymphocytes. J. Clin. Invest. 85, I686-I693. TAMBtJRRINI E., DE LIJCA A., VENTURA G., MAIURO G., SIRACUSANo A., ORTONA E. & ANTINORI A. (i991) Pneumocystis carinii stimulates in-vitro production of tumor necrosis factor-ri by human macrophages. Med. Microbiol. Iminunol. i8o, 15-20. TELZAK E.E., COTE R.J. & GOLI) J.W.M. (1990) Extrapulmonary Pneumocystis carinii infections. J. Infect. Dis. 12, 380-386. VON BEHREN L.A. & PESANTI E.L. (1978) Uptake and degradation of Pneunmocystis carinii by macrophages in vitro. Amt. Rev. Respir. Dis. I I 8, 1051-1059. YONEDA K. & WALZER P.D. (1 980) The interaction of Pneuniocystis carinii with host cells: an ultrastructural study. Inject. Immun. 29, 692703.
J.
Exp. Path. (I991) 72, 589-598
In-vitro interaction of human macrophages with Pneumocystis 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 9 9 I Accepted for publication I 9 June 1 991
Summary. Pneumocystis carinii is an important opportunistic pathogen in patients with compromised cell-mediated immunity. T-cell and macrophage function are believed to be of prime importance in defence against this organism. The present ultrastructural study is aimed at 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 at different intervals and studied by transmission and scanning electron microscopy. The material used for inoculation of macrophages was of identical morphology to previously described P. carinii. When mixed with Pneumocystis in vitro, the macrophages appeared to move towards the organism, extended pseudopods and ingested trophozoites and cysts. Within 24 h, intracellular Pneumocystis underwent progressive degeneration inside macrophage vacuoles. 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 pathogen in patients with impaired cell-mediated immunity and gives rise to life threatening interstitial pneumonia. Originally described by Chagas (I909), P. carinii excited little interest until the first cases of human disease caused by this organism were recognized fifty years later by Deamer and Zollinger (I953). After that, several reports appeared in the literature and in I98I (Gottlieb et al. 198I) Pneumocystis acquired notoriety with its close association
with human immunodeficiency virus (HIV) infection. Despite important advances in the diagnosis (Hopewell I988) and therapy (Kovacs & Masur I988) of Pneumocystis pneumonia, very little is known about the biology, pathogenesis and mechanisms of protective immunity to this organism. Immunosuppressive therapy (Hughes et al. 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, East Birmingham Hospital, Bordesley Green East, Birmingham B9 5ST, UK.
589
590
M. Forte et al.
phage function (Masur et al. I982; Muller et al. 1990) which has previously been suggested as a major defence mechanism against Pneumocystis (Masur & Jones 1978; von Behren & Pesanti 1978). We have studied the interaction between human macrophages and P. carinii using electron microscopy of peripheral blood derived macrophages infected with Pneumocystis in vitro.
Methods Isolation of Pneumocystis carinii Pneuunocystis organisms were obtained from lungs of male Sprague-Dawley rats (HarlanOlac, Bicester, Oxon., UK) which had been immunosuppressed by dexamethasone treatment (2 mg/l in the drinking water) for 8-I o weeks. The animals were killed and the lungs perfused, via the pulmonary artery, with Dulbeccos Modified Eagles Medium (Gibco) prior to homogenization for i min. After filtering through a steel gauze, the homogenate was left to stand for 4 h at 40C in a gravity sedimentation chamber containing a 1-3% gradient of Histopaque (Sigma in Hanks balanced salt solution supplemented with 100 U/ml penicillin/streptomycin (Gibco)). Fractions were collected from the chamber and examined microscopically for Pneumnocystis and rat cell content. The upper fractions were found on average to have ratios of around 3000: I parasite to host cells. The preparation used in subsequent steps contained trophozoites and cysts in a ratio of I10: 1.
Separation of macrophages Defibrinated venous blood was collected from seven healthy adult volunteers from the Department of Immunology at the Medical School, Birmingham, England. Peripheral blood mononuclear cells (PBMC) were separated by density gradient separation on Ficoll/ Hypaque (Pharmacia) (Boyum 1968). These were washed three times in RPMI I 640
(Gibco) and resuspended in RPMI supplemented with glutamine (2 mM), penicillin (I00 ,ug/ml), gentamicin (50 ,g/ml) and io% autologous serum (complete medium). In-vitro infection of macrophages
Isolated PBMC, diluted to io7 cells/ml, were incubated 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 left to adhere to the bottom of the plastic wells or to glass cover slips placed on the bottom of the wells. All non-adherent cells were removed by washing and the media replaced with I ml of fresh complete medium. Approximately i o% of the PBMC adhered as macrophages. The macrophages were inoculated with a I07 suspension P. carinii. The cells were then collected at regular intervals up until 24 h. Macrophages were detached from the bottom of the wells by scraping with a plastic policeman and centrifuged (700 g for 5 min) into a pellet for transmission electron microscopy (TEM). For scanning electron microscopy (SEM), the intact glass cover slip with the adherent cells was used in subsequent steps. Electron microscopy All samples were fixed in 2.5% gluteraldehyde in o.o5 M phosphate buffer pH 7.3 (osmolarity adjusted to 350 mosm with sucrose) for i h. Specimens for TEM were post-fixed in i% osmium tetraoxide in o.o 5% phosphate buffer for i h. Dehydration was carried out in increasing concentrations of acetone for SEM and alcohol for TEM. Material for SEM was critical-point dried in an Emscope CPD75o and sputter coated with approximately 20 nm of platinum in an Emscope SCsoo. Material for TEM was embedded in an Epon/Araldite resin mixture (Mollenhauer I964) and 70-Ioo-nm sections were cut on a Reichert-Jung Ultracut E. The sections were collected on Formvarcoated slot grids and stained with 30% uranyl acetate in methanol for 7 min and
P. carinii and human macrophages
59I
J^~ ~ ~ ~ ~ 1
4t
Fig. i. Pneumocystis carinii used for in-vitro inoculation of macrophages. A, Pneumocystis carinii cyst with internal sporozoites (s) by transmission electron microscopy. Note the tubular structures seen in crosssection adjacent to cyst wall (uranyl acetate and Reynolds lead citrate, x 19 ooo). B, Cyst by scanning electron 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 acetate and Reynolds lead citrate, x 14 ooo; D, platinum coated, x 20 000).
M. Forte et al. 592 Control preparations of macrophages Reynolds lead citrate for 7 min. All speciTem- showed large cells with irregular shaped mens were examined in a Jeol nuclei I 5-20 gim total size. scan. P. carinii organisms isolated from lungs of steroid treated rats were presented in vitro to Kinetics of infection blood-derived human macrophages. Thirty To obtain an estimate number of intra and minutes after inoculation of Pneumocystis the extra-cellular organisms, samples from two macrophages appeared to move towards volunteers were collected at 6, 12 and Pneumocystis with clump formation (Fig. 2a) 24 h. These experiments were performed in and extension of pseudopods towards the parallel with the same antigen preparation. cysts (Fig. 2b, c and d). The pseudopods Sections from those samples were examined appeared to attach preferentially to tubular by TEM and randomly distributed fields were projections rather than the intervening flat photographed at a magnification of x 3ooo. surface of the cysts (Fig. 2d). Macrophages A minimum of five negatives per section also phagocytosed trophozoites by evaginatwere then scored for the numbers of P. carinii ing their cell membrane around the much cysts and trophozoites inside and outside smaller organism cell (Fig. 3). A clear examcells. The number of cells with intracellular ple of this is seen in Fig. 3b where the organisms was also counted. The results macrophage membrane interdigitates with were expressed per IOO cells. the ruffled surface of the trophozoite. This process resulted in most cells showing more than one vacuole containing generally Results cysts, trophozoites, tubular structures and The material isolated from rat lung showed amorphous material (Fig. 3a). Infected maccysts and trophozoites of P. carinii. The cysts rophages, with intracellular cysts at different appeared round, 2-5 gm in diameter, with stages of degeneration, continued to attempt intracystic bodies o. 5-I jim in diameter. The phagocytosis of extracellular organisms. cyst wall, measuring o. I jm on average, was Figure 4 shows the number of organisms formed by three layers: an interior elec- in the first 24 h after in-vitro infection for two tron-dense unit membrane, an intermediate different volunteers. With individual A there electron-lucent layer, and an exterior elec- is an initial increase of extracellular organtron-dense layer, as previously described isms and a reduction in the numbers of (Campbell 9 72; Bedrossian I 989) (Fig. ia). intracellular P. carinii. By I 2 h this pattern is Projections, apparently tubular in the SEM reversed, with an increase in intracellular (Fig. ib), originated from the cyst wall and parasite count and a concomitant reduction appeared in the sections of the TEM (Fig. ib) in extracellular organisms. In the case of as circular structures, 0.05-0.2 jgm in diaindividual B, observations were made only meter, with an outer electron-dense layer up to 12 h. In this case, the numbers of and an electron-lucent centre (Murphy et al. extracellular organisms fell progressively I977). Some of the cysts appeared to have while the intracellular uptake remained released their content and acquired a cressteady at about 30 organisms per IOO cells. cent shape, with trophozoites still closely In all circumstances, the trophozoites count associated (Fig. and d). reflected their initial higher number in relaThe trophozoites, with identifiable nuclei tion to cysts ( Io: i trophozoites to cysts). The in the majority of cases, were polymorphic increase in extracellular organisms seen in with sizes between and 4 jim. The cytoindividual A at I 2 h was due almost exclusiplasm was limited by a single, thick unit vely to an increase in the trophozoites count. membrane which was markedly evaginated The percentage of cells with intracellular (Campbell 1972). organisms (Fig. 5) in individual A showed a ioocx
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variation in keeping with the numbers of extracellular organisms. It started at an initial high value of 36% and fell to a low figure of 8% at I 2 h, rising again after that to 38%. In individual B the percentage of cells with intracellular organisms remained stable around I5% during the I 2 h of the experiment. Six to I 2 h after in-vitro infection of bloodderived macrophages with P. carinii, the number of cells showing phagocytosis increased, resulting in several of them containing one or more vacuoles with organisms in progressive stages of degeneration (Fig. 3c). Extracellular cysts, isolated or in the process of being engulfed by macrophages, could still be identified at 24 h (Fig. 3d). Excess tubular structures which appeared to be derived from protrusions of
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the membrane of Pneumocystis organisms, could be seen in the vicinity of the macrophages at all incubation times. Discussion a well recognized pathogen in immunocompromised patients (Bruke & Good I973). In the overwhelming majority of patients it causes pneumonia that, if untreated, is usually fatal. Recently, several reports have also documented extra-pulmonary pneumocystosis (Telzak et al. I 990). The pathogenesis of P. carinii infection and the mechanisms of host immunity to this organisms are not known at present. Impairment of T-cell (Shellito et al. I990) and macrophage (Masur & Jones I978; von Behren & Pesanti 9 78) function, seem to be major risk factors facilitating infection with
P. carinii is
1
Fig. 3. Later stages of in-vitro infection of macrophages. A, Two hours after inoculation of Pneumocystis carinii. Section of human macrophage with vacuoles containing cysts (c) and collections of rod-like bodies identical to the tubular structures seen emerging from the organism (long arrow). The macrophage is in the 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 of trophozoite (short arrow) (uranyl acetate and Reynolds lead citrate, x 6ooo). C, human macrophage, 1 2 h after inoculation, with several intracellular cysts at different stages of degeneration (uranyl acetate and Reynolds lead citrate, x 3600). D, material collected 24 h after infection, showing a human macrophage with intracellular cysts in the process of ingesting another cyst. Extracellular cysts and trophozoites are still identified (uranyl acetate and Reynolds lead citrate, X 2900).
596
M. Forte et al. this opportunist organism. The particular rived macrophages, in the absence of lymrelevance of the macrophage has been docuphocytes, produce tumour necrosis factor mented in studies showing in-vitro phagoalpha (TNF-a) when exposed to P. carinii in cytosis of Pneumocystis by macrophages from vitro to levels similar to those produced by mice (Masur & Jones 1978) and rats (Von Escherichia coli lipopolysaccharide (LPS) Behren & Pesanti 1978). Biopsy material (Tamburrini et al. I 99 I). This effect could, in from human (Haselton et al. I98I) and rat our system, contribute to the activation of (Yoneda & Walzer I980) lungs has also the monocytes into macrophages. shown intracellular cysts of P. carinii within The organisms were added to human pulmonary macrophages. monocyte-derived macrophages cultured in At present there is no reliable long-term the presence of I0% autologous serum. in-vitro culture system for P. carinii; conse- Subsequently, these cells extended pseudoquently, the best source of antigen remains pods towards pneumocystis (Fig. 2) and by the steroid-treated rat. Although aware of 30 min, intracellular organisms could be antigenic differences between rat and identified. The uptake of P. carinii seemed to human Pneumocystis there is a clear crosslead to degeneration of cysts as several cells reactivity between the antigens of the two could be identified with vacuoles containing organisms (Kovacs et al. I 988) to the point of cysts at different stages of degeneration (Fig. monoclonal antibodies raised to rat Pneumo3c). cystis being used in the diagnosis of PneumoThe kinetics of P. carinii uptake as docucystis pneumonia by immunofluorescence in mented in Figs 4 and 5 reveal a difference bronchoalveolar lavage fluid from immuno- between the two individuals studied. compromised patients (Gill et al. I987). The Although the effect of sampling and countantigen cross-reactivity and the lymphocyte ing can not be completely excluded, the proliferation (Forte et al. I 990) of PBMC from difference may reveal a genuine variability in our volunteers to our antigen preparation the ability of macrophages to handle P. (using a standard 3H-thymidine incorpora- carinii infection. The cells from individual A tion assay (Kumararatne et al. I990)) justify have high levels of uptake initially, with little the use of rat Pneumocystis as a possible apparent destruction of organisms. Indeed, model for host-organism interaction. the total numbers of organisms increased, The present study examines the interac- due to an increase in trophozoites, raising the tion of macrophages and P. carinii by infectpossibility of content release from the cysts. ing blood-derived human macrophages with The increase in total organism numbers and rat Pneumocystis in vitro. The material used the decrease in percentage of cells with for inoculation of macrophage culture (Fig. intracellular organisms proceeds until 12 h i) had an identical morphology to previously when there is a reversal of these trends, described Pneumocystis organisms. In par- possibly corresponding to further activation ticular, the tubular structures seen in associ- of the monocyte-derived macrophages that ation with the cyst wall and the trophozoite are now becoming competent to control membrane (Murphy et al. I977; Haselton et infection. In contrast, individual B shows a ai. I98I), are apparently characteristic of pattern of steady uptake with reduction in this organism. the number of extracellular organisms. The human monocyte-derived macro- Further studies are needed to clarify the phages used in our study were obtained from apparent difference in behaviour of the venous blood of normal volunteers. These blood-derived macrophage from the two had no prior activation other than culture as individuals. plastic-adherent cells for a period of 24 h These results suggest that macrophages before inoculation. It has recently been from normal individuals may be capable of shown that monocytes and monocyte-dedestroying P. carinii without activation by
P. carinii and human macrophages lymphocytes. The interpretation of degeneration was on the basis of disintegration of normal architecture of the cysts seen by electron microscopy, as described by other authors (von Behren & Pesanti 1978). That macrophages kill P. carinii, however, remains presumptive at present in view of the absence of a reproducible method for determining the viability of Pneumocystis in vitro. Also evident was the amount of intra and extra-cellular tubular structures associated with the surface of the organisms as previously reported (Yoneda & Walzer I980). The identity and function of such structures, which seemed to increase over a 24-h period, in the presence of macrophages, is not known, although suggestions of their role in attachment and nutrition have been made previously (Murphy et al. I977; Bedrossian I989). After a 24-h culture period, intracellular, as well as extracellular, intact cysts could still be identified (Fig. 3d). The relatively short time period and the large number of organisms used in the inoculum could account for this persistence. Alternatively, macrophages may need further activation by T-cell products to be completely effective in destroying P. carinii. It seems likely that defence against P. carinii is multifactorial with the main contributors being the T-lymphocytes (Shellito et al. I990), macrophages (Masur & Jones 1 9 78; von Behren & Pesanti I 9 78) and their products (Pesanti I990). Several factors may influence the macrophage attachment and uptake of P. carinii-like antibodies (Masur & Jones 1978) and non-specific receptors, e.g. fibronectin (Pottratz & Martin I990). The influence of several of these factors, the T-cell antigen specific responses, and cytokine production, are under investigation. In conclusion, activated macrophages are required for the destruction of Pneumocystis in vivo. Impaired macrophage function due to factors like steroid therapy or HIV infection, may allow progression of P. carinii infection in the lungs and even result in systemic spread with transport of intracellu-
597
lar viable P. carinii in the blood. These observations highlight the role of the macrophage in host defence against Pneumocystis carinii.
Acknowledgements Dr Miguel Forte was supported by a grant by the Calouste Gulbenkian Foundation, Lisbon, Portugal. The authors wish to acknowledge Professor A.M. Geddes and Dr D. Catty for all their support. References BEDROSSIAN C.W.M. (I989) Ultrastructure of Pneumocystis carinii: a review of internal and surface characteristics. Semin. Diagn. Pathol. 6, 212-237. BOYUM A. (I968) Ficoll Hypaque method for separating mononuclear cells from human blood. Clin. Lab. Invest. 21 (SuPPI. 97), 77-89. BRUKE B.A. & GOOD R.A. (I973) Pneumocystis carinii infection. Medicine 52, 23-51. CAMPBELL W.G. (I 972) Ultrastructure of Pneumocystis in human lung: life cycle of human pneumocystosis. Arch. Pathol. 93, 312-324. CHAGAS C. (i 909) Nova tripanozomiaze humana. Mem. Inst. Oswaldo Cruz I, I59-2I8. DEAMER W.C. & ZOLLINGER H.V. (I 9 5 3) Interstitial 'Plasma Cell' pneumonia of premature and young infants. Pediatrics 12, II-22. FORTE M., PITHIE A., RAHELIJ M., STUBBERFIELD C., KUMARARATNE D. & GEDDES A. (I990) Cytolytic T-cells to Pneumocystis carinii infected macrophages. VI International AIDS Conference, Abstract SA 243. GILL V.J., EVANS G., STOCK F., PARRILO J.E., MASUR H. & KoVAKS J.A. (I987) Detection of Pneumocystis carinii by fluorescent-antibody stain using a combination of three monoclonal antibodies. J. Clin. Microbiol. 25, I837-I840. GOTTLIEB M.S., SCHROFF R. & SCHANKER H.M. (i 98I) Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men. N. Engi. 1. Med. 305, 14251431. HASELTON P.S., CURRY A. & RANKIN E.H. (I.98I) Pneumocystis carinii pneumonia: a light microscopal and ultrastructural study. J. Clin. Pathol. 34, II38-I 146. HOPEWELL P.C. (I988) Pneumocystis carinii pneumonia: diagnosis. J. Infect. Dis. 157, II15III9.
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