Pneumocystis carinii In Vitro A Study by Scanning Electron Microscopy Martin J. Murphy, Jr., PhD, Linda L. Pifer, PhD, and Walter T. Hughes, MD

Pneumocystis carinii is a parasitic microorganism which induces an often fatal pneumonitis in a variety of compromised patients (e.g., premature infants, those with congenital immune deficiency disorders, those therapeutically immune suppressed, etc.). Organisms derived from murine sources were cultivated in vitro on monolayers of primary embryonic chick epithelial lung cells. The infected cultures were then examined by scanning electron microscopy. An examination of the surface ultrastructure revealed pleomorphic organisms which were not only attached to host cells with anchoring fibers but also connected to other parasites via fine fibrils. Another type of fine fibril was observed which may play a role in the organism's nutrition. (Am J Pathol 86:387-402, 1977)

PNEUMOCYSTIS CARPNII, an etiologic agent of pneumonia, is a ubiquitous microorganism associated with significant morbidity and mortality in certain patients. P. carinii pneumonitis occurs in debilitated and premature infants,1-3 occasionally reaching epidemic proportions in nurseries and orphanages, resulting in many deaths.4" Since 1956, when the first case was recognized in North America,7 increased numbers of patients with P. carinii pneumonitis have been diagnosed each year. Pneumonitis caused by this organism compromises infants and children with congenital immune deficiency disorders,,&I patients receiving immunosuppressive therapy for malignant diseases (e.g., leukemia, lvmphoma, multiple myeloma, etc. )1220 or organ transplantation,2' nutritionally deprived infants,3 and patients suffering from certain generalized vascular disorders.' With rare exceptions, infection is localized to the lungs, with pathologic changes confined to alveolar lumina and septa,j9 23 resulting in pulmonary insufficiency. P. catinii is ubiquitous in a variety of mammals,2 and the pneumonitis which it causes may be experimentally achieved in adult rats by repeated From the Infectious Disease Service, St. Jude Children's Research Hospital, Memphis, Tennessee, and the Laboratories of Developmental Hematopoiesis, Sloan-Kettering Institute for Cancer Research, New York, New York. Supported by Grants Al-1 12 7 and CA-08480 from the National Institutes of Health. ALSAC. and by the Jean Shaland Fund. Accepted for publication August 30, 1976. Address reprint requests to Dr. Martin J. Murphy, Jr., Sloan-Kettering Institute for Cancer Research, 410 East 68th Street, New York, NY 10021. 387



American Journal of Pathology

injections with cortisone acetate 24 or may be provoked in rats by dietary deprivation of protein.25 Although Pneumocystis carinii has been known since 1909 when Chagas erroneously identified it as a schizogonic stage of Trypanosoma,26 its exact taxonomy has yet to be established. This, along with the elucidation of its life cycle and infective modus operandi have until of late been frustrated, despite numerous attempts to cultivate it in vitro.23'27'28 We have recently reported that Pneumocystis carinii, derived from human and murine sources, may be cultured in vitro in primary chick epithelial lung (CEL) cells.28 In the reproductive cycle, a vegatative cell, designated trophozoite, attaches by tubular expansions to the host CEL cell and then detaches without entering the cell. Sporozoites develop within the detached young cysts, reaching a maximum number of eight within the mature cyst. Excystment occurs through single or multiple sites in the cyst wall after which the released trophozoite attaches to another host cell.28 Since the CEL cells grow as a monolayer and since P. carinii organisms exclusively reside outside of these host cells, the interaction between parasite and host cell is suited for examination by scanning electron microscopy (SEM) and constitutes the substance of this report. Materials and Methods Details of the cultivation of Pneumocystis carinii, their morphology by light microscopy, and the kinetics of proliferation in vitro and incorporation of tritiated nucleotides and amino acids have been reported.28 Inocula

P. carinii organisms used to inoculate the CEL cell cultures were obtained from lung washings of Sprague-Dawley rats which had been treated with cortisone acetate to provoke infection.29 Lungs from healthy rats, examined and found to be free of P. carinii, were used to prepare a "mock" inoculum for control cultures. Cell Cultures

Embryonic chick epithelial lung (CEL) cells were prepared from 14-day-old embryonated eggs by successive treatments with 0.25% pronase.30 The aggregates of epithelial cells were counted and resuspended in medium 199 containing 10% fetal calf serum and antibiotics. The CEL cultures were established in Leighton tubes containing several sterile mica discs (12 mm diameter) and were maintained at 35 C for 24 hours prior to P. carinii or mock inoculation. At the time of inoculation, CEL monolayers were 75 to 90% confluent and contained no more than 10% fibroblasts. Scanning Electron Microscopy The mica discs bearing the attached infected CEL cells were withdrawn from the cultures between 4 and 38 hours after inoculation and processed for SEM. Uninfected CEL cell cultures prepared in the same manner but using normal rat lung washings as a mock inoculum served as a control. The mica discs were rinsed briefly with a gentle stream

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of chilled saline, then fixed bv total immersion in chilled 4% glutaraldehyde in 0.1 NM phosphate buffer at pH 7.4 for 2 hours. The discs were then successively transferred into two petri dishes containing 0. M phosphate buffer, followed immediately by immersion in 1 % osmic acid buffered by 0.2 M phosphate (pH 7.4) for 1 hour at 4 C, and finally washed in two changes of double distilled water. Dehydration was accomplished with increasing concentrations of ethanol to absolute alcohol. The mica discs were then transferred to a 1: 1 solution of absolute alcohol and propylene oxide for 30 seconds, followed by immersion in 100% propylene oxide for 15 seconds, after which thev were removed to air with virtually instantaneous drning.3' The mica discs, cemented to aluminum SEM stubs with an electroconductive glue (Aquadag), were rotary coated at 0l torr with 50 A of carbon and 100 X of palladium and then examined at 20 or 25 KVP at various angles to the incident electron beam in one of the following scanning electron microscopes: Philips SEM-500, Zeiss Novascan 30, or AMR-IO00A.

Results Nomedature

The nomenclature used to describe the various stages in the life cycle of Pneumocystis carinii differs among authors.24.32,&¶ We shall use the following terms, based on the size of the organism: Sporozoite refers to membrane-bounded intracystic bodies and those in the process of being released from the cyst. Those "budding" from the cvst measure an average of 1 i in diameter. Trophozoite refers to the extracystic form from the point of detachment from the cyst and is therefore smaller than the cyst, measuring an average of 3 A in diameter. Cyst refers to pleomorphic cells which may be round, oval, or with single or multiple surface concavities. They average 5.5 g in diameter, with a range of 4 to 8 A in diameter. Transmission electron microscopic observations of control uninfected CEL cell cultures, inoculated with washings from normal rat lungs (i.e., mock-infected), revealed a monolayer of cells with surfaces characterized bv microvilli, tight intercellular junctions, and prominent round protuberances (diameter approximately 0.5 A) representing underlying membrane-bounded vesicles and mitochondria.* Control cultures, therefore, were composed of monolayers of epithelial cells with no other attached surface structures and, as will be shown, were distinct from similar CEL cultures inoculated with P. carinii (Figures 1-8). Scanning electron microscopy of CEL cell cultures inoculated with P. carinii 4 and 8 hours earlier are depicted in Figures 1 and 2. The P. carinii organisms not only adhere to the host monolayer but appear to be connected to the CEL by fine filaments (Figure IA). Some of these filaments are contiguous with underlying host cells, while others are freelv suspended except for their attachment to the parasite and the CEL.



American Journal of Pathology

The pleomorphic structural characteristics of P. carinii are readily apparent, ranging from round organisms with a smooth surface-tentatively identified as trophozoites (Figure 1A)-to crinkled cysts with microvilli dotting the external wall (Figures iB, 2, and 7B). The surface undulations may be slight (Figure 2A) or appear as a well-developed concavity (e.g., Figure 4D). An extreme concavity is represented in Figure 2B. An en face thin section of such a cyst, encompassing the concavity, could in the transmission electron microscope (TEM) appear crescent or cup-shaped, a form frequently observed in the TEM.20,2833,34 This stage of P. carinii is almost devoid of microvilli by comparison with the external cystic wall of organisms having moderate undulations. P. carinii organisms are not only visualized individually (Figures 1 and 4) but also in aggregate (Figures 2, 3, and 7). The aggregated (agglutinated?) parasites are as diverse in surface morphology as are their individual counterparts (Figure 4). One feature of the aggregated forms, however, is remarkable. Groups of P. carinii are frequently associated with an arabesque, membrane-bounded matrix (Figures 3 and 7A). Whether this structureless material is of cyst or host cell origin is undetermined. The material is evident both free of CEL cells, as well as contiguous with the underlying CEL cells. Another aspect in the multifaceted complexion of P. carinii is an array of fibrils coursing between individual organisms (Figure 5). These fibrils are generally more slender than the fibers joining P. carinii to the underlying host cells. An interparasitic fibril which traverses a CEL cell and bifurcates while attached to a cyst is shown at high magnification in Figure 8. In addition to fibers which appear to anchor P. carinii to host cells and those fibrils which communicate between parasites, there are tenuous fibrils which clearly touch host cells (Figure 6) and appear to anastamose with the CEL cell plasmalemma (Figure 6B and C). The size of the fibrils at the point of attachment is 0.15 to 0.20 ,u in diameter; they emanate from isolated P. carnnii already anchored to a host cell or from a parasite which is in aggregate with other P. carinii. Figure 4C and D exemplifies a morphologic aspect of P. carinii which, although not a feature of every cyst examined, is one which is frequently observed. "Buds" are seen protruding from attached cysts which may represent sporozoites in the process of excystment. Discussion

The scanning electron microscope affords a valuable adjunct to the morphologic examination of biologic material. Its advantages of magnifi-

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cations greater than those obtainable with light microscopy and a depth of focus unavailable with TEM are of considerable importance. Caution, however, must be taken with tissue preparation so that artifacts of technique are also not "magnified" and misinterpreted as facts.31'35 In this present study we have heeded these precautions and monitored our preparations by bright field, phase contrast, and Nomarski differential interference light microscopy, as well as conventional TEM. No organisms described as Pneunocystis carinii were observed in control CEL cell cultures treated with mock inocula from normal rats. The organisms under discussion were originally derived from P. cariniiinfected rat lungs. Our observations in vitro demonstrate that P. carinii adhere to lung epithelial cells, confirming other in vivo ultrastructural studies." Despite their intimate contact, however, the parasites did not invade the host cell en masse, confirming our TEM observations." Indeed, intracellular parasitism in natural hosts only occurs within phagocytic alveolar macrophages.20 Previous authors, using the TEM, have outlined a putative life-cycle for P. carinii describing the size and ultrastructural anatomy of cysts, trophozoites (or trophic stages) and sporozoites (or intracystic bodies)."'-' When viewed with the SEM, the broad size range of organisms makes it difficult, if not arbitrary, to define a point at which the trophozoite becomes a cyst. This is of particular note since the organisms in culture were not proliferating synchronously and represented both growth and dormant stages of the parasite.2' Emphasis will therefore be ascribed to P. carinii structure and not whether a given structure is representative of cysts or trophozoites. One of the most prominent surface features of P. carinii is the array of fibers and fine fibrils. Basically, three types have been encountered: a) interparasitic fibers (Figures 2A, 5, and 7), b) fibers which appear to anchor the parasite to the host cell (Figure IA), and c) fine fibrils common to both the parasite and host cell (Figure 6). Barton and Campbell ' originally interpreted the "tubular expansions" as anastomosing membranes, later calling them "filopodia" or "pseudopodia"M and on this basis suggested that many of the organisms were motile. Our current studies viewing static organisms in the SEM neither support nor negate such an interpretation. It is of interest, however, that thin sections of these fibers reveal a tubular ultrastructure, with an electron-lucent core surrounded by an electron-opaque perimeter." Such a configuration might not only provide support or anchorage but offer a conduit for the exchange and/or procurement of material. We have demonstrated that P.



American Journal of Pathology

carinii organisms have the distinct opportunity for interparasitic exchange of protoplasm. In concordance with other studies,24'28'33 we have no indication that any form of the organism is phagocytic or enters host cells. The observations of Barton and Campbell,24'37 as well as those of our own,28 indicate that P. carinii metabolizes low-molecular-weight substances. This is now given morphologic credence. Morphologic evidence suggests that the fibrillar structures (e.g., Figure 6) may function as tubules through which the organism obtains its essential nutrients. The alveolopathy produced by P. carinii in vivo is marked by desquamative alveolitis, with intraalveolar accumulation of macrophages, cellular debris, and P. carinii. The attendant functional pulmonary insufficiency may result in hypoxemia with a respiratory rate which may reach 80 to 100 respirations/min. '20 Examples of aggregated P. carinii in vitro illustrated in Figures 2, 3, and 7 are comparable to clustering seen by light microscopy from infected lungs. In addition, a flocculent material with a ruffled surface is also evident (Figures 3 and 7A), although its origin is not yet defined. It is likely that some of this material may represent degenerating epithelial cells in view of the in vitro cytopathic effects of the CEL cells observed between 24 and 72 hours after inoculation with P. carinii. 28

Vavra and Kucera state that "it is not quite understood what the mechanism is that allows the intracystic bodies (sporozoites) to get out of the cyst."33 Candidate sporozoites in the process of excystment are illustrated in Figure 4C and 4D. Consequent to the loss of intracystic sporozoites, the cyst wall collapses (Figure 4D), at times leaving a deep invagination (Figure 2B). If such a cyst were thinly sectioned at right angles to the plane of the cleft, a crescent-shaped structure would result. Such sickleshaped cysts are a common feature of earlier studies using the TEM 20,28,33,34

While it is not within the purview of this present report to advance the definitive thesis on the taxonomic classification of P. carinii, a note regarding its identity and relatives is in order. That Pneumocystis carinii is eucaryotic and unicellular in nature and hence belongs to the subkingdom Protista is a common ground for agreement. From this point on, however, there is considerable debate. Protistans are divided among a) Protozoa and b) Fungi. Vavra and Kucvera conclude that Pneumocystis is not a protozoan and that there is no good reason for excluding it from the fungi, and tentatively place it in class Ascomycetes, subclass Hemiacomycetidae. On the other hand, Ham et al., in accord with Campbell,38 consider these organisms to be motile and most likely protozoan rather than fungal.3'

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The advent of techniques with w%hich Pneumocystis carinii max be reliablv propagated in vitro, coupled with ultrastructural, biochemical and serologic investigations, may not only solve the classification of this parasite but, more importantly, help to resolve the nature of the infection in man. References 1. Huneycutt HC, Anderson WR, Hendrv WS: Pneumocystis carinui pneumonia: Case studies with electron microscopy. Am j Clin Pathol 41:411-418, 1964 2. Robbins JB: Pneumocystis carinii pneumonitis: A review. Pediatn Res 1:131-158, 1967 .3. Dutz W\T: Pneumocystis caninii pneumonia. Pathol Annu 5:309-341, 1970 4. Vanek J, Jirovec 0, Lukes J: Interstitial plasma cell pneumonia in infants. Ann 5. 6.

7. 8. 9.

10. 11.

12. 13. 14.

13. 16. 17. 18. 19. 20.


Pediatr 180:1-21,-1953

Lim SK, Moon CS: Studies on Pneurnocystis carinii pneumonia. II. Epidemiological and clinical studies of 80 cases. Jonghap Med (Korea) 6:774-6, 1960 Hyun BH, Varga CF, Thalheimer LJ: Pneunocystis carinui pneumonitis occurring in an adopted Korean infant. JAMA 195:784-786, 1966 Dauzier G, Willis T, Bamett RN: Pneumocystis carnii pneumonia in an infant. Am J Clin Pathol 26:787-793, 1956 Lingeman CH, Schulz DM, Lukemyer JW%: Pneumocystis carinii pneumonia in congenital rubella. Am J Dis Child 113:585-587, 1967 Salfelder K, Schwartz J: Pneumocystosis: Current concepts and recent advances. Am J Dis Child 114:693-699, 1967 Barnett RN, Hull JG, Vortel V, Kralove H, Schwartz J: Pneumocystis carinii in lymph nodes and spleen. Arch Pathol 88:175-180, 1969 Walzer PD, Schultz MG, Western KA, Robbins JB: Pneumocystis carinji pneumonia and primary immune deficiency diseases of infancy and childhood. J Pediatr 82:416-422, 1973 Ruskin J, Remington JS: Pneunocystis carinii infection in the immunosuppressed host. Antimicrob Agents Chemother 7:70-76, 1967 NMulligen WJ, Krause FD, Morningstar W, Baum GL: Pneunocystis cannii infestation: Four cases with associated lymphoma. Ohio State NMed J 64:1017-10,20, 1968 Devita VT, Emmer NI, Levine A, Jacobs B, Berard C: Pneunocystis carinii pneumonia: Successful diagnosis and treatment of two patients with associated malignant processes. N Engl J Med 280:287-291, 1969 Moore DL, Carnahans CE, Mills SD, Burgert EO: Pneumocystis carinii pneumonitis, complicating leukemia. NMayo Clin Proc 44:162-168, 1969 Goodell B, Jacobs JB, Powell RD, Devita VT: Pneumocystis carnnii: The spectrum of diffuse interstitial pneumonia in patients with neoplastic diseases. Ann Intern Med 72:337-340, 1970 Luna MA, Bodev GP, Goldman ANM, Lichtiger B: Pneumocystis carinii pneumonitis in cancer patients. Texas Rep Biol Med 30:41-56, 1972 Sedaghation MR, Singer DB: Pneumocystis carinii in children with malignant disease. Cancer 29:772-777, 1972 Hughes WT, Price RA, Kim HK, Coburn TP, Grigsby D, Feldman S: Pneumocystis carinii pneumonitis in children with malignancies. J Pediatr 82:404-415. 1973 Price RA, Hughes WT: Histopathology of Pneumocystis cannii infestation and infection in malignant disease in childhood. Hum Pathol 5:737-752, 1974 Hamburger J, Crosnier J, Dormont J: Experience with 45 renal homotransplantations in man. Lancet 1:985-992, 1965



American Journal of Pathology

22. Robbins JB: Immunological and clinicopathological aspects of Pneumocystis carinii pneumonitis. Birth Defects 9:219-244, 1968 23. Sheldon WH: Pulmonary Pneumocystis carinii infection. J Pediatr 61:780-791, 1962 24. Barton EG Jr, Campbell WG Jr: Pneumocystis carinii in lungs of rats treated with cortisone acetate: Ultrastructural observations relating to the life cycle. Am J Pathol 54:209-236, 1969 25. Hughes WT, Price RA, Sisko F, Havron WS, Kafatos AG, Schonland M, Smythe PM: Protein calorie malnutrition: A host determinant for Pneumocystis carinii infection. Am J Dis Child 128:44-52, 1974 26. Chagas C: Nova tripanozomiaza humana: Estudos sobre a morfologia o ciclo evolutivo do Schizotrypanum cruzi n. gen. n. sp. agente etiologio de nova entidade morbida do homen. Mem Inst Oswaldo Crus Rio 1:159, 1909 27. Gajdusek DC: Pneumocystis carinii: Etiologic agent of interstitial plasma cell pneumonia of premature and young infants. Pediatrics 19:543-565, 1957 28. Pifer LL, Hughes WT, Murphy MJ Jr: Propagation of Pneumocystis carinii in vitro. Pediatr Res (In press) 29. Kim HK, Hughes WT, Feldman S: Studies of morphology and immunofluorescence of Pneumocystis carinii. Proc Soc Exp Biol Med 141:304-309, 1972 30. Darlington RW, Portner A, Kingsbury DW: Sendai virus replication: An ultrastructural comparison of productive and abortive infections in avian cells. J Gen Virol 9:169-177, 1970 31. Murphy MJ Jr: Effects of vitamin A on the erythrocyte membrane surface. Blood 41:893-899, 1973 32. Murphy MJ Jr, Pifer LL, Hughes WT: Unpublished observation 33. Vavra J, Kucera K: Pneumocystis carinii Delanoi: Its ultrastructure and ultrastructural affinities. Protozoology 17:463-483, 1970 34. Ham EK, Greenberg SD, Reynolds RC, Singer DB: Ultrastructure of Pneumocystis carinii. Exp Mol Pathol 14:362-372, 1971 35. Murphy MJ Jr, Morris B: A study of the peritoneal surface of the diaphragm by stereoscan electron microscopy. Proceedings of the Seventh Congress of the International Society for Electron Microscopy, Vol 3. 1970, pp 587-588 36. Murphy MJ Jr: The shape of blood platelets: An application of lyophilization and scanning electron microscopy. Thromb Diath Haemorrh 28:237-243, 1972 37. Barton EG Jr, Campbell WG: Further observations on the ultrastructure of Pneumocystis. Arch Pathol 83:527-534, 1967 38. Campbell WG Jr: Ultrastructure of Pneumocystis in human lung: Life cycle in human pneumocystosis. Arch Pathol 93:312-324, 1972

Acknowledgments The authors acknowledge the valuable technical assistance of Diane Woods and are especially grateful to J. D. Rogers of Philips Electronics, Inc., Dr. Ulrich Kumpf of Carl Zeiss, Inc, and George Bruno of AMR Corporation for the use of their scanning electron microscopes.


1A-mDryonic chicGk epiuieal lung (CEtL) cell

culture 4 nours after inoculation with Pneu-

mocystis carinii (Pc) illustrating fibers (asterisk) anchoring P. carinii to the CEL cells (350 angle, x 2430). B-CEL cells 8 hours after inoculation with P. carinii (Pc) with examples of two cysts (390 angle, x 9000).


Figure 2A-CEL cells 4 hours after inoculation with P. carinii (Pc) demonstrating an aggreate of the organism, some with interparasitic fibrils (asterisk) (300 angle, x 5000). B-Aggregate of P. carinii in CEL cell culture which had been infected 4 hours earlier. One deeply invaginated cyst is indicated by an arrow. (350 angle, x 9300).





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Figure 4A-CEL cell culture 18 hours after inoculation with P. carinii (Pc) in which a cyst remains attached to what may be the degenerate CEL cell. (350 degree, x 5000). B-P. carinii cyst at 8 hours after initial infective inoculum (350 angle, x, 5370). C and D-P. carinii cysts at 4 hours after initial infective inoculum; Arrows indicate presumptive sporozoltes during excystment (45.50 angle, C, x 7500; D, x 8900).


5A and B-P. carinii Fqe parasites (arrow) as well

(400 angle, A,


4 hours after initias inoculum illustrating fibrils which course between as those which communicate between the organisms and host cells (arrow)

9530, B,




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q .




Figure 6A-CEL cell culture 8 hours after inoculation with P. carinii exemplifying presumptive nutritive fibrils (arrows) (300 angle, x 5830). B and C-High magnifications of the fibrils showing the anastamosis of host and parasite plasmalemmae (300 angle, x 1850).



m S } ~~~~A


which is characteristic of a large Fei 7A-CEL cell culture 24 hours after inoculation with P. carinii The area within the matrix

aggregate of parasites interspersed with a flocculent, amorphic (arrows). rectangle is magnified in BL (450 angle, x 2560) B-Higher magnification of A illustrating the surface P. x undulations and microvillar structures typical of carinii (45° angle, 12,780).

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Pneumocystis carinii in vitro: A study by scanning electron microscopy.

Pneumocystis carinii In Vitro A Study by Scanning Electron Microscopy Martin J. Murphy, Jr., PhD, Linda L. Pifer, PhD, and Walter T. Hughes, MD Pneum...
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