Plasmodium sporozoite-host cell interactions during sporozoite invasion* J.P. Vanderberg & M.J. Stewart

In vitro and in vivo studies were performed to clarify the nature of some interactions between Plasmodium berghei sporozoites and rodent host cells. Videomicroscopic observations were made on in vitro inter- actions between sporozoites and cultured host cells (rodent peritoneal , W138 human lung fibroblasts, and HepG2 human hepatoma cells). The results showed a diversity of dynamic interactions and sporozoite activities, including active sporozoite penetration, often followed by sporozoite escape, killing of invaded macrophages, ofsporozoites by macrophages, and elaboration ofa weblike structure by the sporozoite, following its escape from cells. Other studies were performed to examine host cell inflammatory responses to challenge sporozoites in the of rats that had been previously immunized with radiation-attenuated sporozoites. The results showed a variety of focal inflammatory infiltrates in the livers ofthe challenged animals. The relevance ofthese observations to the problems ofsporozoite invasion into naive and immunized animals is discussed.

Introduction eral of these factors act together against the pre- A Plasmodium sporozoite that reaches the circulatory erythrocytic stages of the parasite. system ofits mammalian host must distinguish among The purpose ofthis paper is to review some of the a succession of host cells if it is to be successful in ways in which sporozoites interact with mammalian ending up as a pre-erythrocytic schizont (PES). First, host cells, and to present some new data describing the sporozoite, while flowing rapidly through the interactions between the parasite and host cells circulation, must stop in the and become firmly in vitro and in vivo. attached to one of the two types of cells lining the lumen of the hepatic sinusoid: Kupffer cells or endo- Sporozoite interactions with hepatic thelial cells. Then, it must pass through one of these sinusoidal cells cells into the space of Disse and recognize and invade its final destination, the . It is impressive to Sporozolte binding within the hepatic sinusoid: consider that the very same sporozoite must be pro- Kupffer cells vs. endothelial cells grammed to recognize and invade at least two distinct types of host liver cells. We will show that sporozoites Sporozoites flowing through the of a mamma- are aggressive in their ability to penetrate these cells. lian host rapidly penetrate the liver, many of them During this journey, the sporozoite must run a apparently in a single passage (1). In a well-adapted gauntlet of host defence cells before it can impact host-parasite system, the large majority of sporo- upon its final destination within the liver. Some sporo- zoites successfully invade the liver, few appear to be zoites are successful in reaching and invading hep- strained out and sequestered in other capillary beds of atocytes; others are intercepted during their travels the body. The evidence for this includes the fact that through the body, or are destroyed within the liver only several sporozoites are needed to induce an as they are developing into PES. To develop better infection in well-adapted simian malaria systems and strategies to intercept or destroy the parasite during that as many as 95% of the P. berghei sporozoites any of these stages, it behoves malariologists to under- injected into their natural tree rat host may develop stand how the parasite interacts with host cells and into PES (2). This strongly implies a liver-specific how host defence mechanisms function against the mechanism for sporozoite entrapment. It is important parasite in nonimmunized and immunized hosts. to recognize that cells within the blood have no direct Sporozoite-induced immunity is a multifactorial contact with . Blood flowing through the phenomenon; protective immunity occurs when sev- liver sinusoids has contact only with the cells that line the sinusoids. An important unanswered question relating to * From the Department of Medical and Molecular Parasitology, sporozoite biology is whether a sporozoite, rapidly New York University School of Medicine, 550 First Avenue, New flowing through a , interacts with the York, NY tO016, USA. Correspondence to Dr. J.P. Vanderberg. liver via a sporozoite ligand-liver receptor interaction

74 Bulletin of the World Health Organization, 68 (Suppl.): 74-79 (1990) Plasmodlum sporozolte-host cell interactions and remains there, or whether a nonspecific sinusoidal static carcinoma cells, which move through endo- trapping mechanism filters out sporozoites as they thelial fenestrae to invade the liver parenchyma (11). pass. In either event, the attachment would require The early anatomists recognized that endothelial strong binding between the sporozoite and a cell cells lining blood vessels of the liver are structurally lining the liver sinusoid to prevent the sporozoite different from those lining blood vessels in other parts from being swept away in the sinusoidal blood flow. of the body, and that the liver cells are permeable to There are only two candidates for this: Kupffer cells blood components that could not penetrate else- and endothelial cells. where. It is now recognized that these cells contain a number of receptors designed to remove components Posible role of host plasma proteins in medating destined for the liver, and that the two classes of cells recognition. Though the circumsporozoite (CS) pro- lining the sinusoid have different types of receptors. tein covering the sporozoite is a prime candidate for For instance, endotoxin is taken up almost entirely by a ligand that could recognize a sinusoid-lining cell, Kupffer cells (12), whereas the endothelial cells have a proteins binding to sporozoites could high specificity for acetylated low-density lipoprotein also play a role in mediating this putative recognition. (13), transferrin (14), bound albumin (12) and heparin A serum glycoprotein binds to P. berghei and (12). P. knowlesi sporozoites and appears to enhance sporozoite binding to macrophages in vitro (3, 4). Dynamk Interacffons betwen sporozoltes and Furthermore, incubation of sporozoites in serum macrophages In vitro prior to injecting them intravenously into their mam- In view of the possible role of liver macrophages in malian hosts has been shown to greatly enhance their mediating infection by sporozoites (as described infectivity (reviewed in (3)). above), in intercepting and killing some sporozoites attempting to invade the liver, and in possibly func- Posaibie roie of the Kupfer ceii. The suggestion that tioning as antigen-presenting cells during the induc- sporozoites are phagocytosed by Kupffer cells prior to tion ofthe immune response, many workers have been infection of hepatocytes has long been entertained interested in studying sporozoite- inter- (5, 6). This concept is attractive from an evolutionary actions in vitro. standpoint because sporozoites of reptilian and avian These studies have involved incubating sporozo- malarias invade and then develop in reticuloendo- ites with target host cells under given conditions for a thelial cells, including macrophages. The proposal has given period of time, after which the slide is fixed, recently been revived (7, 8). These workers have ar- stained, and viewed through a microscope. Unfortu- gued that there is a nonspecific initial interaction nately, this approach results in a frozen picture of the between sporozoites and Kupffer cells, followed by interaction at an arbitrary point in time. Our own active Kupffer cell phagocytosis of the sporozoite. recent studies, using videomicroscopy, have shown Our own data have shown, instead, active and aggres- that there is a diversity of dynamic interactions be- sive penetration of macrophages by sporozoites tween sporozoites and host cells that is not recog- in vitro (see below). A relevant question is whether the nizable on fixed slides. We used Plasmodium berghei rapidly moving sporozoites attach to Kupffer cells by sporozoites obtained from the salivary glands of in- nonspecific mechanisms, or whether this attachment fected Anopheles stephensi mosquitos, and pipetted is enhanced by CS protein or by opsonization of the drops of a sporozoite suspension on coverslips con- circulating sporozoite by plasma proteins. taining monolayers of peritoneal macrophages from mice (Swiss-Webster) and rats (Norway brown). The medium consisted of Minimal Essential Medium Po"sibe role of the sinusoidal-finIng endothelial cell. (MEM) with 3% bovine serum albumin and all pre- Because ofthe speed with which sporozoites can leave parations were maintained at 37°C. Observations the blood to invade hepatocytes, we suggested a rapid were made by phase-contrast microscopy, using a and direct passage through the endothelial cells (9). video camera and a video cassette recorder to record Others have argued that this is impossible because of observations. the small diameters of the fenestrae that pass through results demonstrated the wide these cells (8). However, our demonstration of rapid, Our following active invasion by sporozoites through cells (including range of sporozoite-macrophage interactions: liver endothelial cells) in vitro indicates that sporo- A. No interaction. The most common observation zoites could actively squeeze through these contrac- was that actively gliding sporozoites would come in tile, microfilament-enveloped, narrow openings (10) repeated contact with macrophages, without either of or even pass through intact cells. Similar passage of the cells showing any discernible interaction. Even relatively large cells has been demonstrated for meta- when there was active filipod ruffling on the part of

WHO Bulletin OMS: Supplement Vol. 68 1990 75 J.P. Vanderberg & M.J. Stewart the macrophage, the sporozoite often repeatedly Sporozoite interactions with glided over the macrophage and then moved away. B. External killing by the macrophage. Some sporo- hepatocytes zoites glided over the macrophage, then stopped and Once sporozoites have passed through the wall of the apparently were killed and deformed over about a liver sinusoid and entered into the space of Disse, the 20-minute period, while the sporozoites remained final step of the invasion process can take place, attached to the surface of the macrophage. namely, attachment to and invasion of hepatocytes. C. Phagocytosis. Some sporozoites were actively This is not as formidable a problem for the sporozoite phagocytosed by the macrophage over a 5-20-minute as its prior invasions; sporozoites are aggressively period, and were seen to remain within the sporozoite invasive to start with; once they are in the sluggish even after observation for up to one hour. circulation of the space of Disse, strong binding affin- D. Phagocytosis with sporozoite escape. Often a ities are not needed to keep them from being swept sporozoite in the process of being phagocytosed away. Indeed, the fact that sporozoites invade a wide would escape by gliding away, generally leaving be- variety of non-hepatic cell types in vitro (15) shows hind an extended macrophage filipod, which would that hepatocyte-specific receptors are not essential. then contract back into the macrophage. Much of the work on hepatocyte receptors has been E. Active penetration. The most dramatic event done by Hollingdale et al. (review in (16)), who pro- observed was active penetration by sporozoites into posed that a peptide region aminoterminally adjacent macrophages (even into activated macrophages). This to the tandem repeats of the CS protein binds to process took only a few seconds and was accom- specific receptors on the hepatocyte. This group has panied by vigorous thrashing and flexing on the part also developed in vitro invasion models consisting of of the sporozoite. Sometimes the sporozoites would W138 human embryonic lung fibroblasts and HepG2 remain inside the macrophage. Often, however, they human hepatoma cells. Our own studies have con- would emerge from inside. It was not uncommon to firmed the validity of these findings and the usefulness see a sporozoite repeatedly enter and exit a macro- of these cell lines in studying invasion by P. berghei phage in a "needling" fashion. sporozoites (17). F. Macrophage killing. Macrophages that had We have recently extended our in vitro video- been penetrated usually exhibited a cytoplasmic microscopic studies to these cell lines, using the same "blebbing" at the site of sporozoite entry or exit. procedures that we previously used for our studies This normally led to macrophage death within a few with macrophages. Our most significant finding has minutes. been that only rarely was there evidence of a dra- G. Sporozoite "tether" and "web" formation. After matic sporozoite-host cell interaction upon contact. emergence from a macrophage, sporozoites often had Sporozoites often came in repeated contact with the long, thick "tethers" trailing behind them. These had host cell surface, each time gliding away and return- considerable tensile strength, sometimes allowing ing. Other sporozoites glided directly on the surface of sporozoites to pull around an entire cell, while being host cells for several minutes at a time before gliding attached via the "tether". The "tethers" were also away. Actual penetration of the host cell, when it elastic or contractile in nature, sometimes contracting occurred, was either accompanied by a pronounced and pulling back a sporozoite that had stretched it flexing of the sporozoite or by an apparently effortless out. Often, a meshwork of "tethers" was laid down, so gliding of the sporozoite into the cell. Such sporozo- that it looked as though the sporozoite had spun an ites often continued gliding within the confines of a cell elaborate web. or even passed from cell to cell, sometimes leaving a H. Inhibition of sporozoite motility by anti-sporo- clear "wake" behind them within the cytoplasm of the zoite antibodies. Addition of monoclonal antibody host cells being traversed. Sporozoites that exited host directed against the immunodominant repeating epi- cells often emerged with "tethers" similar to those seen tope of the CS protein caused a rapid immobilization attached to sporozoites emerging from macrophages. of sporozoites and the formation of a CSP (CS pre- cipitation) reaction at the trailing end of the sporo- zoite. Some sporozoites tended to move extremely Defensive host cell responses to slowly for a minute or two after their main forward progress had been stopped; they appeared to leave a sporozoites in vivo trail of CSP reaction behind them as they moved Cellular reactions around bursting PES in the livers forward. Active invasion by sporozoites was never of monkeys infected with P. cynomolgi were first observed in preparations that received antibody. reported by Shortt & Garnham (18). Garnham & Such sporozoites were slowly phagocytosed by Bray (19) pointed out that in haemosporidiids, the macrophages. parasite developing inside a tissue cell provokes no

76 WHO Bulletin OMS: Supplement Vol. 68 1990 Plasmodlum sporozolte-host cell Interactions cellular reaction. Once it has reached maturity and sporozoites developed a range of inflammatory re- the schizont has ruptured, there is an immediate sponses; these were more numerous in the sporozoite- inflammatory response. Such responses have been immunized rats, but were observed to some degree observed in rodent malaria: P. berghei (20-22), and P. even in na*ve rats challenged with sporozoites. These chabaudi and P. yoelii (23). responses included granulomatous reactions (associ- However, a mononuclear infiltrate attacking ap- ated with lymphocytes, but without the notable proximately 30% ofintrahepatocytic PES within nor- presence of foreign body giant cells), periportal infil- mal brown Norway rats has recently been described tration (with abundant ), Kupffer cell (24). In view of these interesting findings, we at- hyperplasia (primarily mid-zonal), and small, dense tempted to repeat this study with the same type of rats aggregates of lymphocytes. The latter appeared to be of a similar age. Our results have shown no such the most notable response in the sporozoite-immun- inflammatory responses, except around bursting PES. ized rats. The fact that there appeared to be more It would be of interest to determine the experimental inflammatory foci than the numbers of sporozoites differences responsible for these dissimilar findings by used for challenge makes it impossible at this point to our two laboratories. Such information could lead to associate a given type of response with a specific a better understanding of the factors that influence a cytotoxic action against challenge sporozoites or the host inflammatory response against PES. young pre-erythrocytic forms that developed from An inflammatory response against the parasite in them. Some of the PES that developed in sporozoite- the livers of sporozoite-immunized animals has long immunized rats appeared to have no inflammatory been entertained as a possibility. More than 20 years cells closely associated with them, whereas others ago we observed granulomatous reactions in the livers were surrounded by a histiocytic response. of mice that had been immunized with irradiated P. berghei sporozoites, then challenged with normal sporozoites (Vanderberg and Nussenzweig, unpub- lished observations). However, it was difficult to Conclusions follow up this finding at that time because the sporo- zoites used were heavily contaminated with bacteria Dynamic Interactions between sporozoltes and and mosquito debris; control mice immunized with host cells In vitro noninfected salivary glands had similar, though fewer, It is quite clear that our procedure demonstrates a inflammatory foci in their livers. diversity of dynamic interactions that can occur be- More recently, we have readdressed the problem tween sporozoites and host cells. Such interactions by use of an immunization procedure that does not in have been unrecognizable by previous approaches, itself produce background liver pathology, namely, which consisted of microscopic examination of allowing sporozoite injection via the bites of irradi- stained slides fixed at arbitrary times during in vitro ated, infected mosquitos (25). Norway brown rats incubation. The key point of our findings is that the were immunized by gamma-irradiating mosquitos in- sporozoite cannot be considered as though it were fected with P. berghei sporozoites and allowing the merely an affinity bead with surface ligand molecules; mosquitos to feed on the anaesthetized rats. Two such it is a dynamic, motile organism with a complex feedings, 15 days apart, produced a suboptimal pro- repertory of activities. tection, permitting us to study the fate of the small We have shown that sporozoites have the ability percentage of sporozoites that evaded the humoral to move in and out of macrophages, destroying them response and transformed into mature PES. Rats in this process. The relevance of these in vitro observa- were challenged intravenously with 106 sporozoites tions to what actually occurs in vivo with in situ each, 15 days after the final immunization and liver Kupffer cells remains to be determined. Previous biopsy specimens were taken 40, 44 or 48 hours post- studies with fixed in vitro preparations of peritoneal challenge to study the histopathology. The immunized macrophages reported that macrophages that were rats had high anti-sporozoite antibody titres and found to have sporozoites within them for periods developed on the order of 5% of the PES found in longer than 30 minutes had become morphologically non-immunized control rats; the numbers found in altered and destroyed (26). Furthermore, an in vivo these immunized rats (approximately 5 PES per cm2 study (27) reported apparent morphological altera- of liver tissue section) were sufficient to study the fate tion of a Kupffer cell adjacent to a hepatocyte that of these "breakthrough" schizonts. had been penetrated by a sporozoite, thus raising the Immunized animals that were not subsequently possibility that the sporozoite had moved through the challenged did not develop hepatic inflammatory Kupffer cell. reactions recognizably different from those of naive The relative infrequency of clear interactions be- nonimmunized rats. However, all rats challenged with tween motile sporozoites and target host cells (HepG2

WHO Bulletin OMS: Supplement Vol. 68 1990 n7 J.P. Vanderberg & M.J. Stewart and WI38) that they are known to invade under the form. This may occur in naive animals under certain same in vitro conditions is puzzling. Other workers conditions (24) or in animals that have been immun- have proposed an interaction between a ligand con- ized with irradiated sporozoites, as has been reported sisting of an epitope of the CS protein on the in this paper. sporozoite and a putative receptor on the hepatocyte In view of the rapidity with which sporozoites are surface (16, 28). However, our in vitro observa- able to leave the blood and invade hepatocytes, cellu- tions failed to detect any clear attachment or lar inflammatory responses by themselves are unlikely recognition by the great majority of sporozoites that to be of significant value in intercepting circulating came in contact with the host cells; sporozoites re- sporozoites. Of course, sporozoites that have been peatedly glided over the surface of these cells and then opsonized by antibody may be more likely to be glided away. This does not fit the classical concept of phagocytosed and destroyed by Kupffer cells (as we specific binding between a ligand and a cell surface have shown for peritoneal cavity macrophages); receptor, followed by a recognizable biological modi- activation of Kupffer cells by immunization might fication of the target cell. It also further supports our enhance this process even further. The most signific- contention that hepatocyte-specific receptors are not ant actions of the inflammatory cells that we have essential for normal invasion by sporozoites. described would likely be against parasites that have Our results also support our previous proposal already invaded host cells. This could be due either that the CSP reaction is a result of antibody pre- to the production of IFN-gamma, which could act cipitation of a normal flow of CS protein down the against PES within hepatocytes, or by the direct pellicle of the moving sporozoite (29-31). This pre- cytotoxic action ofthese inflammatory cells. However, cipitation of the CS antigen is associated with a rapid there is at present no evidence that any of these inflam- inhibition of the main forward movement of the matory infiltrates are around parasites within hepato- sporozoite. Such an inhibition of circulating sporo- cytes. Some of them could well be associated with zoites in vivo would prevent them from actively invad- Kupffer cells that have internalized sporozoites, and ing cells lining the liver sinusoid; this would be suffic- might well even represent a stage of antigen pre- ient to explain the protective effects ofanti-CS protein sentation by Kupffer cells to surrounding lympho- antibody in vivo (17). Accordingly, if antibody can act cytes. Clearly, future immunohistochemical studies in this way to prevent sporozoites from even reaching will be necessary to specifically associate the parasite the space of Disse, any subsequent hypothetical action with these inflammatory foci and to determine what of the antibody in inhibiting sporozoite interaction type of liver cell the parasite is within. with a putative hepatocyte surface receptor would be Finally, the significance of a histiocytic infiltrate irrelevant to functional antibody activity in vivo. around some of the PES that were able to develop in the immunized animals is unclear. This appears to be the first report describing an inflammatory response around PES even before the first PES begin to mature Defensive host cell responses to sporozoltes and release merozoites. These inflammatory re- In vivo sponses were seen as early as 40 hours after sporozoite The major problem with exclusive dependence upon injection, whereas blood transfers from these rats to antibody-mediated immunization against sporozoites naive rodents showed that the first infective pre- is that it must act very quickly and be 100% effective. erythrocytic merozoites did not appear in the blood Some sporozoites can penetrate the liver within min- until more than 44 hours after sporozoite injection. utes after inoculation, and thus may be able to escape Whether these inflammatory cells represent a func- antibody assault by rapidly passing from the blood tional immune response against some developing PES into hepatocytes. Unfortunately, even a single escaped or whether they represent inflammatory cells that sporozoite can induce a blood infection with have arrived in response to already damaged or dead concomitant disease. PES remains to be seen. It would be useful if the immune system had a "second shot" at the parasite, either in the form of enhanced phagocytosis of circulating sporozoites, killing ofthe developing PES within the hepatocyte or an enhanced phagocytosis of merozoites leaving a Acknowledgements burst PES. There is now evidence that gamma-inter- We thank Serena Chew for help in performing some of the feron can act against PES developing within the videomicroscopic observations and Dr J.A. Terzakis for hepatocyte (32). There is also evidence that the host consultation on liver histopathology. Grant support: U.S. may mount an inflammatory cell response that could Public Health Service, NIAID grants A124528 (J.P.V.) and act directly against the developing pre-erythrocytic A124615 (M.J.S.)

78 WHO Bulletin OM8: Supplement Vol. 68 1990 Plasmodlum sporozolte-host cell Interactions

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