EXPERIMENTAL

PARASITOLOGY

14,

228-231 (1992)

RESEARCH

BRIEF

Plasmodium fragile: Cytoadherence of Parasitized Rhesus Monkey Erythrocytes to Human Endothelial Cells under Shear Flow Conditions TIMOTHY School of Chemical Engineering

M.

WICK

AND

VALERIE

and Cellular Biomechanics Laboratory, Atlanta, Georgia 30332-0100, U.S.A.

LOUIS Georgia Institute

of Technology,

WICK, T. M., AND LOUIS, V. 1992. Plasmodium fragile: Cytoadherence of parasitized rhesus monkey ervthrocvtes to human endothelial cells under shear flow conditions. Experimental Parasitology j4, 228-23 1.

In human Plasmodium falciparum malaria, severe pathology is associated with cytoadherence of parasitized erythrocytes to cerebral microvascular endothelium leading to vessel occlusion, tissue necrosis, and possibly severe neurological symptoms (Toro and Roman 1978; MacPherson et al. 1985; 00 et al. 1987). Cytoadherence is limited to erythrocytes infected with late-stage parasities (trophozoites and schizonts) (Luse and Miller 1969, 1971; MacPherson et al. 1985) and is related to expression of parasite-derived antigens on the infected erythrocyte surface (Kilejian 1979; Leech et al. 1984; Howard et al. 1990). In vitro, red cells parasitized with P. falciparum schizonts and trophozoites rosette uninfected erythrocytes (Udomsangpetch et al. 1989; Handunnetti et al. 1989; Hasler et al. 1990) and adhere to cultured endothelial cells (Udeinya et al. 1981; Ockenhouse and Chulay 1988; Wick and Louis 1991); apparently by different mechanisms (Hasler et al. 1990; Kaul et al. 1991). In ex viva perfusion studies, rosette formation, in combination with endothelial cytoadherence, amplifies postcapillary venule occlusion as compared to cytoadherence alone (Kaul et al. 1991). Primate infection with P. fragile bears significant similarity to human P. falciparum malaria and has been proposed as an animal model for human P. .falciparum malaria (Fremount and Miller 1975; Chin et al. 1979; David et al. 1988). Most significantly, only erythrocytes containing late-stage parasites sequester in the microcirculation of various organs (Fremount and Miller 1975; David et al. 1988) and cytoadherence appears to predominate in the venules (Fremount and Miller 1975). However, unlike human P. falciparum malaria, P. fragile infection does not result in significant cerebral sequestration (Fremount and Miller 1975). Erythrocytes infected with late-stage P. fragile parasites express electron-dense knobs which are the sites of endothelial cell attachment in viva (Fremount and Miller 1975). In vitro, simian erythrocytes infected

with late-stage P. fragile parasites can also form rosettes (David et al. 1988). These data suggest that the mechanism of parasitized erythrocyte sequestration and microvascular obstruction are similar in human P. falciparum and simian P. fragile malarias (David et al. 1988; Udomsangpetch et al. 1991). However, little is known about the mechanism of P. fragile-infected erythrocyte adherence to endothelial cells or its relationship to microvascular occlusion. Therefore, cytoadherence of P. .franiZe-infected rhesus monkey erythrocytes to human endothelial cells was quantified under postcapillary venule shear stress conditions in a parallel-plate flow chamber. P. fragile parasites were collected from rhesus monkeys (Maraca mulatta) with patent P. fragile infection and cultured in rhesus monkey erythrocytes (Chin et al. 1979) under candle jar conditions (Jensen and Trager 1977). During subculture, parasites were not cloned to select for specific adherence properties. Just prior to an adherence assay, parasitized erythrocytes containing predominantly schizonts and trophozoites were washed and resuspended to 1% hematocrit in serum-free medium (SFM; consisting of Medium 199, supplemented with 5.0 (*g/ml bovine insulin, 5.0 kg/ml human transfertin, 0.2% human albumin, 0.1 mg/ml streptomycin and penicillin, and 0.292 mg/ml L-glutamine). Human umbilical vein endothelial cells were harvested by collagenase digestion (JatTe et al. 1973) and passaged into single-well LabTek chambers (Wick and Louis 1991). At confluence, endothelial cell monolayers were assembled in a parallel-plate flow chamber (chamber gap width = 250 urn) and adherence under venous shear flow conditions was visualized in real time. Briefly, endothelial cell monolayers were rinsed for 5 min with SFM to remove serum and adhesive proteins secreted during culture. Parasitized or uninfected erythrocytes suspended in SFM were then perfused over the monolayer for 10 min. Nonadherent

228 0014-4894/92 $3.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any foml reserved.

ENDOTHELIAL

CYTOADHERENCE

erythrocytes were rinsed from the chamber by a lomin perfusion with SFM at the same shear stress. Adherent erythrocytes were enumerated in 20 random microscopic fields and normalized to adherent red blood cells per square millimeter of endothelium (RBC/mm’). A single-classification analysis of variance was utilized to differentiate between adherence of parasitized and uninfected erythrocytes for each experiment (Morrison 1983). At 1.0 dyne/cm* shear stress, cytoadherence of P. fragile-infected RBC to human umbilical vein endotheha1cells ranged from 8.3 f 0.5 to 21.1 + 1.3 RBC/mm* (mean -C SEM) (Table 1) and was 2.5 to 83-fold greater than cytoadherence of uninfected erythrocytes (P < 0.01 for each experiment). Cytoadherence was dependent upon erythrocyte infection with schizonts and trophozoites (Table 1). Ring-infected erythrocytes did not cytoadhere in agreement with other observations that only erythrocytes infected with P. fragile schizonts and trophozoites sequester in vivo (Fremount and Miller 1975; David et al. 1988) and form rosettes in vitro (David et al. 1988). Furthermore, adherent erythrocytes were attached as singlets. Erythrocyte clusters or rosettes were not observed, either adhering to the endothelium or flowing in the chamber. To determine whether shear stress influenced the amount of cytoadherence, P. fragile-infected erythrocyte adherence to endothelial cells was quantified at 0.5, 1.0, 1.5, and 2.0 dynes/cm2 (Fig. 1). At 0.5 dyne/cm*, parasitized erythrocyte adherence was 19.0 * 3.5 RBC/mm’ and was significantly greater (P < 0.01) than uninfected erythrocyte adherence at 0.5 dyne/cm2 (5.0 ? 2.3 RBC/mm*). Increasing shear

Adherence of P. fragile

229

I 0.50

1.0

1.5

2.0

Shear Stress (dynes/cm’) FIG. 1. Cytoadherence of Plasmodium fragileinfected erythrocytes to endothelial cells is a function of shear stress. Data are means -+ SEM for four experiments performed with endothelial cells, parasitized erythrocytes, and uninfected erythrocytes from the same batch culture. Adherence of parasitized erythrocytes was significantly decreased at each step increase in shear stress (P < 0.01, except for adherence of parasitized erythrocytes at 1.0 dyne/cm* vs. 1.5 dynes/cm*, P < 0.05). Adhesion of parasitized erythrocytes was greater than adherence of uninfected erythrocytes at each shear stress studied (P < 0.01). stress to 1.O, 1.5, or 2.0 dynes/cm* decreased parasitized erythrocyte adherence to 14.1 + 2.6, 11.5 2 2.8, or 7.3 5 3.3 RBC/mm2, respectively (P < 0.05 for each step increase in shear stress). At the equivalent shear

TABLE I Parasitized and Uninfected Erythrocytes to Human Microvascular Endothelial Cells at 1.O dyne/cm*

Adherent RBC/mm* Parasitized erythrocytes

Uninfected erythrocytes

21.1 2 8.3 + 20.0 k 15.1 + 9.9 f 8.6 2

0.5 k 0.5 0.1 + 0.1 3.7 k 0.7 3.2 2 0.5 1.5 -c 0.2 3.4 f 0.7ll

1.3 (5) 0.5 (4) 1.5 (2) 1.0 (2) 0.9 (2) 1.2 (1)

OF P. fragde

(5) (2) (2) (2) (2) (1)

Percentage parasitemia 2.5 1.0 5.0 2.5 2.0 1.0

Percentage late-stage parasitemia ++ + ++++ ++ + +

Percentage rings

Fold increase

5 -

42.5 83.0 5.4 4.7 6.6 2.5

Note. Values shown are means 5 SEM of P. fragile-infected erythrocyte adherence per square millimeter of endothelial surface area. The value in parentheses indicates the number of endothelial monolayers studied for each determination in a given experiment. For each experiment (row), endothelial cells and parasitized erythrocytes were from identical cultures. For each endothehal slide tested, flow was continuous and constant for the duration of the experiment. Perfusion media was changed by means of a three-way stopcock. Adherence of parasitized erythrocytes was significantly greater than adherence of uninfected erythrocytes in each experiment (P < 0.001, except llP i 0.01). The relative number of rings and late-stage parasites present in the erythrocyte suspensions was classified as: - , (0.5%; +, approximately 0.5%; +, approximately 1%; + +, l-2%; + + +, 2-3%;or ++++,>3%.

230

WICK AND LOUIS

stress, uninfected erythrocyte adherence was 2.4 2 1.4, 2.8 2 1.9, and 3.1 2 2.2 RBC/mm’, respectively, and significantly less than parasitized erythrocyte adherence (P < 0.01). Shear stresses in the postcapillary venules are on the order of 1.O dyne/cm’ and are significantly lower than those in the capillaries or precapillary arterioles (Chien 1972; Karino and Goldsmith 1987).The data of Fig. 1 indicate that P. fragile-infected erythrocyte adherence is likely maximal in the postcapillary venules as has been demonstrated for P. falciparum (Raventos-Suarez et al. 1985;Wick and Louis 1991).This is in agreement with histologic data demonstrating P. fragile sequestration in rhesus monkey venules (Fremount and Miller 1975) and suggests that hemodynamic forces contribute to the localization of cytoadherence in viva. In addition to infection with late-stage parasites, in vitro rosetting requires 50% serum (David et al. 1988). To avoid the simultaneous appearance of rosette formation and endothelial cytoadherence (and the associated difficulties in quantifying adherence), the present experiments were performed in the absence of serum. Thus, these data likely represent basal levels of cytoadherence under postcapillary venule shear conditions. Even under these conditions, P. fragileinfected erythrocytes were 2.5- to 83-fold more adhesive than uninfected erythrocytes (Table I), suggesting that endothelial adherence is an inherent property of parasitized erythrocytes. Rosettes were not observed under the conditions of the present assay. In the presence of plasma in vivo, endothelial cell cytoadherence and/or rosette formation are likely increased, leading to additional sequestration and microvascular obstruction (Kaul er nl. 1991). Do mixed species adherence studies such as those reported here have relevance to P. fragile malaria in vivo? While this question cannot be answered directly, cytoadherence of P. falciparum-infected erythrocytes to human endothelium in vitro and to rat endothelium ex vivo demonstrate important similarities. Specifically, in both systems, only parasitized erythrocytes adhere and cytoadherence is dependent upon infection with late-stage parasites (Udeinya ef al. 1981; Raventos-Suarez er al. l985), adherence is strong enough to withstand postcapillary venule blood shearing forces (Raventos-Suarez et al. 1985; Wick and Louis 1991), and adherence is inhibited by anti-CD36 antibodies (Kaul ef al. 1991; Oquendo et al. 1989). More importantly, no specific differences have been reported. Therefore, it appears that endothelial cytoadherence is a property of sequestering Plasmodium parasites. The present data demonstrate specific binding of P. fragileinfected rhesus monkey erythrocytes to human endothelial cells, suggesting that this system can be utilized for additional mechanistic studies of cytoadherence. The data obtained will then likely be relevant to cytoadherence and sequestration in vivo, and because

animal studies are more feasible than those involving humans, in vitro inhibitors of cytoadherence can be analyzed in monkeys in vivo. P. fu/ciparum-infected human erythrocytes have previously been shown to adhere to human umbilical vein endothelial cells (Udeinya et al. 1981). Furthermore, endothelial adherence is dependent upon erythrocyte infection with schizonts or trophozoites (Udeinya et al. 1981), occurs in the absence of serum or exogenously added adhesive proteins (Wick and Louis 1991), and is strong enough to withstand postcapillary venule shearing forces (Wick and Louis 1991). Similar observations are reported here utilizing P. fragile-infected erythrocytes. These data suggest that the mechanism of P. fragile and P. falciparum cytoadherence to endothelial cells is similar. In the absence of rhesus monkey endothelial cultures, future cytoadherence studies utilizing human endothelial cells will likely have relevance to simian P. fragile malaria and possibly by extension to human P. fulciparum malaria (Fremount and Miller 1975; Chin et al. 1979; David et al. 1988). (Human umbilical vein endothelial cells were a kind gift of Dr. Thomas Lawley, Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia. The receipt of parasitized erythrocytes from Dr. Phuc Nguyen-Dinh, Centers for Disease Control, Malaria Branch, Atlanta, Georgia, is gratefully acknowledged. The authors are also indebted to Dr. Pascal Millet for his critical evaluation of this manuscript. This work was supported by the Biomedical Research Support Grant Committee of Georgia Institute of Technology.) REFERENCES CHIEN, S. 1972. Present state of blood rheology. In “Hemodilution: Theoretical Basis and Clinical Application” (H. Schmid-Schbnbein and K. Messmer, Eds.), pp. l-45. Karger, Basel. CHIN, W., Moss, D., AND COLLINS, W. E. 1979. The continuous cultivation of Plasmodium fragile by the method of Trager-Jensen. American Journal of Tropical Medicine

and Hygiene 28, 591-592.

DAVID, P. H., HANDUNNETTI, S. M., LEECH, J. H., GAMAGE, P., AND MENDIS, K. N. 1988. Rosetting: A new cytoadherence property of malaria-infected erythrocytes. American Journal of Tropical Medicine and Hygiene 38, 289-297.

FREMOUNT,H. N., AND MILLER, L. H. 1975. Deep vascular schizogony in Plasmodium fragile: Organ distribution and ultrastructure of erythrocytes adherent to vascular endothelium. American Journal of Tropical Medicine and Hygiene 24, l-8. HANDUNNETTI, S. M., DAVID, P. H., PERERA, K. L. R. L., AND MENDIS, K. N. 1989. Uninfected erythrocytes form “rosettes” around Plasmodium

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falciparum infected erythrocytes. American Journal of Tropical Medicine and Hygiene 40, 115-l 18. HASLER, T., HANDUNNETTI, S. M., AGUIAR, J. C., VAN SCHRAVENDUK, M. R., GREENWOOD, B. M., LALLINGER, G., CEGIELSKI, P., AND HOWARD, R. J. 1990. In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients. Blood 76, 1845-1852. HOWARD, R. J., HANDLJNNETTI, S. M., HASLER, T., GILLADOGA, A., DE AGIJIAR, J. C., PASLOSKE, B. L., MOREHEAD, K., ALBRECHT, G. R., AND VAN SCHRAVENDUK, M. R. 1990. Surface molecules on Plasmodium falciparum-infected erythrocytes involved in adherence. American Journal of Tropical Medicine and Hygiene 43, 15-29. JAFFE, E. A., NACHMAN, R. L., BECKER, C. G., AND MINICK, C. R. 1973. Culture of human endothelial cells derived from umbilical veins: Identification by morphologic and immunologic criteria. Journal of Clinical Invesligaiion 52, 2745-2756. JENSEN, J. B., AND TRAGER, W. 1977. Plasmodium falciparum in culture: Use of outdated erythrocytes and description of the candle jar method. Journal of Parasitology 63, 883-886. KARINO, T., AND GOLDSMITH, H. L. 1987. Rheological factors in thrombosis and haemostasis. In “Haemostasis and Thrombosis” (A. L. Bloom and D. P. Thomas, Eds.), 2nd ed., pp. 739-755. Livingstone, Edinburgh. KAUL, D. K., ROTH, E. F., JR., NAGEL, R. L., HowARD, R. J., AND HANDUNNETTI, S. M. 1991. Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions. Blood 78, 812-818. KILWIAN, A. 1979. Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum. Proceedings of the National Academy of Sciences USA 76, 4650-4653. LEECH, J. A., BARNWELL, J. W., AIKAWA, M., MILLER, L. H., AND HOWARD, R. J. 1984. Plasmodium falciparum malaria: Association of knobs on the surface of infected erythrocytes with a histidinerich protein and the erythrocyte skeleton. Journal of Cell Biology 98, 1256-1264. LUSE, S. A., AND MILLER, L. H. 1969. Distribution of mature trophozoites and schizonts of Plasmodium falciparum in the organs of aotus trivirgatus, the night monkey. American Journal of Tropical Medicine and Hygiene 18, 860-865. LUSE, S. A., AND MILLER, L. H. 1971. Plasmodium falciparum malaria: Ultrastructure of parasitized erythrocytes in cardiac vessels. American Journal of Tropical Medicine and Hygiene 20, 655-660. MACPHERSON, G. G., WARRELL, M. J., WHITE,

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N. J., LOOAREESUWAN, S., AND WARRELL, D. A. 1985. Human cerebral malaria: A quantitative ultrastructural analysis of parasitized erythrocyte sequestration. American Journal of Pathology 119, 385-401. MORRISON, D. F. 1983. “Applied Linear Statistical Methods,” pp. 280-293. Prentice-Hall, Englewood Cliffs, NJ. OCKENHOUSE, C. F., AND CHULAY, J. D. 1988. Plasmodium falciparum sequestration: OKM5 antigen (CD361 mediates cytoadherence of parasitized erythrocytes to a myelomonocytic cell line. Journal of Infectious Diseases 157, 584-588. 00, M. M., AIKAWA, M., THAN, T., AYE, T. M., MYINT, P. T., IGARASHI, I., AND SCHOENE, W. C. 1987. Human cerebral malaria: A pathological study. Journal of Neuropathology and Experimental Neurology 46, 223-23 1. OQUENDO, P., HUNDT, E., LAWLER, J., AND SEED, B. 1989. CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes. Cell 58, 95-101. RAVENTOS-SLJAREZ, C., KAUL, D. K., MACALUSO, F., AND NAGEL, R. L. 1985. Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes. Proceedings of rhe National Academy of Sciences. USA 82, 3829-3833. TORO, G., AND ROMAN, G. 1978. Cerebral malaria: A disseminated vasculomyelinopathy. Archives of Neurology 35, 271-275. UDEINYA, I. J., SCHMIDT, J. A., AIKAWA, M., MILLER, L. H., AND GREEN, I. 1981. Falciparum malaria-infected erythrocytes specifically bind to cultured human endothelial cells. Science 213, 555557. UDOMSANGPETCH, R., BROWN, A. E., SMITH, C. D., AND WEBSTER, H. K. 1991. Rosette formation by Plasmodium coatneyi-infected red blood cells. American Journal of Tropical Medicine and Hygiene 44, 395L401. UDOMSANGPETCH, R., WAHLIN, B., CARLSON, J., BERZINS, K., TORII, M., AIKAWA, M., PERLMANN, P., AND WAHLGREN, M. 1989. Plasmodium falciparum-infected erythrocytes form spontaneous erythrocyte rosettes. Journal of Experimental Medicine 169, 1835-1840. WICK, T. M., AND LOUIS, V. 1991. Cytoadherence of erythrocytes to Plasmodium falciparum-infected human umbilical vein and human dermal microvascular endothelial cells under shear conditions. American Journal of Tropical Medicine and Hygiene 45, 578-586. Received 3 July 1991; accepted with revision ber 1991

21 Octo-

Plasmodium fragile: cytoadherence of parasitized rhesus monkey erythrocytes to human endothelial cells under shear flow conditions.

EXPERIMENTAL PARASITOLOGY 14, 228-231 (1992) RESEARCH BRIEF Plasmodium fragile: Cytoadherence of Parasitized Rhesus Monkey Erythrocytes to Human...
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