343

Cytotoxic activity of 13-hydroxyoctadecadienoic acid against Toxoplasma gondii W. R. HENDERSON JR 1 and E. Y. CHI 2 Departments of ^Medicine and ^Pathology, University of Washington, Seattle, WA 98195, USA (Received 6 December 1991; revised 3 April 1992; accepted 3 April 1992) SUMMARY

Recent data indicate that platelets may play an important role in the host defence against Toxoplasma gondii infections. T. gcmdzY-stimulated human platelets release thromboxane A 2 (TXA 2 ) and 12-hydroxyeicosatetraenoic acid (12-HETE) from arachidonic acid and 13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid (Yong et al. 1991 ; Henderson et al. 1992). We have previously demonstrated that t h e eicosanoid TXA 2 has potent cytotoxic activity against T. gondii trophozoites (Yong et al. 1991). In this study, we examined whether 12-HETE, 13-HODE, and linoleic acid also have toxoplasmacidal activity. 13-HODE at concentrations ^ 10"8 M rapidly induced cytotoxic changes in T. gondii. Ultrastructural changes induced by 13-HODE in T. gondii included an initial leakage of cytoplasmic contents into a space between the inner and outer parasite bilayer membrane units which was followed by intracellular vacuolation and loss of cytoplasmic contents. In contrast, linoleic acid and 1 2 - H E T E lacked toxoplasmacidal activity at 10~10-10~6 M concentrations. These data indicate that 13-HODE, a product of linoleic acid metabolism, has potent cytotoxic activity against T. gondii; this toxoplasmacidal activity may be important in the inflammatory response to this pathogen. Key words: Toxoplasma gondii, cytotoxicity, 13-hydroxyoctadecadienoic acid (13-HODE), linoleic acid, 12-hydroxyeicosatetraenoic acid (12-HETE), thromboxane A2 (TXA 2 ).

INTRODUCTION

A variety of host defence mechanisms operate against the protozoan Toxoplasma gondii. Polymorphonuclear and mononuclear phagocytes (Wilson & Remington, 1979; Murray et al. 1985), interferon-yactivated macrophages (Suzuki et al. 1988), and natural killer (NK) cells (Hatcher & Kuhn, 1982) each exert toxoplasmacidal activity. Recent attention has been directed to the role of platelets in the host defence against T. gondii (Ridel et al. 1988; Yong et al. 1991). Platelets may interact with T. gondii during the parasitaemia which occurs frequently in acute and chronic T. gondii infections in animals and humans (Verlinde & Makstenieks, 1950; Beverley, 1959; Remington, 1961; Siegel et al. 1971; Raizman & Neva, 1975). T h e development of vasculitis, thrombosis, and coagulation necrosis in tissues (e.g. placenta, brain, eyes, skin and kidneys) infected by T. gondii (Remington & Desmonts, 1990) also suggests that platelets may play an inflammatory role in these infections. Studies by Ridel et al. (1988) have demonstrated that T. gondii tachyzoites are killed by IgE-bearing platelets obtained from rats infected by T. gondii. Further, infusion of these platelets into nu/nu Fischer rats which are highly susceptible to T. gondii infection provides significant protection against T. gondii (Ridel et al. 1988). These data indicate that in immunocompromised rats, platelets are effector cells which limit parasite dissemination in T. gondii infections.

We have recently reported that human platelets exert potent cytotoxic activity against T. gondii (Yong et al. 1991). Platelets were found to adhere to the surface of T. gondii and induce lysis of surface membranes and cytoplasmic contents of the parasites. Human platelet-mediated toxoplasmacidal activity was unaffected by either non-immune or immune serum. T. gonrfn-stimulated platelets released the arachidonic acid (5, 8, 11, 14-eicosatetraenoic acid) metabolite thromboxane (TX)A 2 , and this release of thromboxane was important in the cytolytic process. In addition to thromboxane, human platelets also release monohydroxy-derivatives of both arachidonic acid and linoleic acid (9, 12-octadecadienoic acid) after interaction with T. gondii (Henderson et al. 1992). These lipid mediators are the arachidonic acid metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid derivative, 13-hydroxyoctadecadienoic acid (13H O D E ) (Henderson et al. 1992). We report here that 13-HODE has potent cytotoxic activity against tachyzoites of T. gondii. In contrast, its precursor fatty acid, linoleic acid, as well as 12-HETE, lacks toxoplasmacidal activity. MATERIALS AND METHODS

Materials 13(S)-HODE [13(S)-hydroxy-9(Z), ll(E)-octadecadienoic acid], 12(S)-HETE [12(S)-hydroxy-5(Z), 8(Z), 10(E), 14(Z)-eicosatetraenoic acid], and linoleic

Parasitology (1992), 105, 343-347 Copyright © 1992 Cambridge University Press 24-2

W. R. Henderson and E. Y. Chi

344

60

TO

c CO .c o o

oxide, and Medcast resin from Ted Pella, Inc. (Redding, CA); osmium tetroxide from Stevens Metallurgical Corp. (New York, N Y ) ; sodium cacodylate trihydrate from Scientific Chemical Co. (Huntington Beach, CA); and paraformaldehyde from J. T. Baker Chemical Co. (Phillipsburg, NJ). All other reagents used were of the highest commercial grade available.

40

X

o o c>

20

Parasite culture

Linoleic acid

13-HODE

12-HETE

Fig. 1. Morphometric analysis of the effect of linoleic acid, 13-HODE, and 12-HETE on Toxoplasma gondii. T. gondii tachyzoites were incubated in PBS in the absence or presence of linoleic acid, 13(S)-HODE, or 12(S)-HETE at the indicated concentrations for 30 min at 37 °C. The reaction mixtures were then prepared for transmission electron microscopy and the percentage distribution of cytotoxic changes (as compared to no cytotoxic changes) of T, gondii was determined by morphometric analysis as described in the Materials and Methods section. Percentage cytotoxic changes above the background release of 8'7 + l-9% (mean + s.E.; n = 6) are shown. A total of 3986 T. gondii tachyzoites was randomly selected and examined in these studies. ( • ) 10"10 M; (H) 10-8 M; ( 0 ) 10"6 M.

T. gondii (RH strain) tachyzoites were maintained by intraperitoneal passage in Balb/c mice as previously described (Locksley, Fankhauser & Henderson, 1985; Yong et al. 1991). T h e organisms were harvested in Dulbecco's phosphate-buffered saline (PBS; GIBCO Laboratories, Grand Island, NY) that was Ca 2+ /Mg 2+ -free and filtered through a 3 /tm polycarbonate filter (Nucleopore Corp., Pleasanton, CA) to exclude mononuclear and polymorphonuclear phagocytes and cellular debris. After centrifugation at 1000^ for 15 min at 4 °C, the organisms were washed twice and resuspended in PBS containing Ca 2 + /Mg 2 + . Trypan blue dye exclusion testing indicated that more than 95 % of the isolated T. gondii were viable. Ultrastructural studies

P< 0-001

10

30

60 Time (min)

Fig. 2. Kinetics of 13-HODE-induced Toxoplasma gondii cytotoxic changes. T. gondii tachyzoites were incubated in PBS in the absence (O) or presence ( # ) of 10"6 M 13(S)-HODE for 0-90 min at 37 °C. Percentage cytotoxic changes of the organisms was determined by morphometric analysis as described in the Materials and Methods section. Probability values for the difference from T. gondii alone are shown where significant (P < 005). acid [9(Z), 12(Z)-octadecadienoic acid] were obtained from Cayman Chemical Co. (Ann Arbor, M I ) ; potassium ferryocyanide from Sigma Chemical Co. (St Louis, M O ) ; glutaraldehyde, propylene

T. gondii were incubated in PBS with the reaction mixture supplements as indicated in the legends to the figures, centrifuged at 1000 £ for 15 min, and fixed in 4 % glutaraldehyde and 2 % paraformaldehyde in 0-1 M sodium cacodylate buffer, pH 7-4 for 4 h. The samples were washed 3 times in 0-1 M cacodylate buffer, and post-fixed with 1 % osmium tetroxide and 1 % potassium ferrocyanide in 0-1 M cacodylate buffer for 4 h. After washing 3 times in double-distilled water, the samples were enblocstained with 0'5 % uranyl acetate for 20 min, washed 3 times with double-distilled water, embedded in 2 % agar, chilled, and cut into 6-9 x 1-0 m m 3 blocks. The samples were dehydrated in a graded series of alcohol washes, treated 3 times each for 30 min with propylene oxide, infiltrated with Medcast resin for 18 h under vacuum, and then embedded in Medcast resin. Three 1-0 mm 3 blocks were randomly selected and thick sections were cut at 1-0 /an. T w o of these blocks had thin sections cut at approximately 70 nm with a diamond knife (Diatome Ltd, Bienne, Switzerland). A ribbon of 20-30 thin sections was placed on a 200-mesh grid coated with 1 % Parlodion (Mallinckrodt, St Louis, MO). The grids were examined using a JEOL 100B electron microscope (Japan Electron Optics Laboratory, Tokyo, Japan) at 60 kV as previously described (Henderson et al. 1980).

Toxoplasmacidal activity of 13-HODE

Fig. 3. For legend see p. 346.

345

W. R. Henderson and E. Y. Chi

Morphometry Morphometric analysis was performed as previously described by Weibel (1979) to determine the distribution of morphological changes induced in T. gondii after incubation with 13-HODE, linoleic acid or 12-HETE. The T. gondii were categorized as either normal in appearance or exhibiting at least one of the following cytotoxic changes: (a) extravasation of cytoplasmic contents between the inner and outer surface membranes, (b) surface membrane vesicle formations, (c) extracellular loss of cytoplasmic contents or (d) intracellular vacuolation. For morphometric analysis, all transmission electron micrographs were taken at a magnification of x 7000. Ten randomly selected fields in each grid were photographed.

RESULTS

Since 13-HODE and 12-HETE are released by platelets after interaction with T. gondii (Henderson et al. 1992), we examined whether these compounds exert toxoplasmacidal activity. Exogenous 13(S)HODE, when incubated with the organisms for 30 min at concentrations > 10~8 M, caused cytotoxic changes as determined by transmission electron microscopy (Fig. 1). Approximately 50% of the organisms exhibited cytotoxic changes above control levels after incubation with 10"6 M 13(S)-HODE. In contrast, T. gondii ultrastructure was unaffected by either its precursor fatty acid, linoleic acid or 12(S)HETE over the same concentration range (Fig. 1). 13-HODE-induced T. gondii cytotoxicity was rapid in onset with approximately 40 % of the organisms exhibiting cytotoxic changes higher than control after 10 min of incubation (Fig. 2). The morphological changes in the T. gondii induced by 10~6 M 13-HODE are illustrated in Fig. 3. T. gondii are crescent-shaped protozoa (Fig. 3 A) which are surrounded by two closely apposed, separate bilayer membrane units (Fig. 3B). Incubation of 10"6 M 13(S)-HODE with T. gondii for 10 min resulted in bulging of the outer parasite bilayer membrane secondary to leakage of cytoplasmic contents through the inner parasite bilayer mem-

346

brane structure (Fig. 3C). By 30 min (Fig. 3D), 13-HODE induced extensive disruption of surface membrane structures, intracytoplasmic vacuolization, and loss of cytoplasmic contents of T. gondii.

DISCUSSION

In this study, we have demonstrated a novel cytotoxic effect of 13-HODE (Js 10~8 M) against T. gondii. 13-HODE-induced damage of T. gondii was characterized by an initial leakage of cytoplasmic contents into a space between the inner and outer separate bilayer membrane units of the organisms. This was followed by extensive disruption of the surface membrane structure with loss of intracellular contents of T. gondii. Human platelets induce similar cytotoxic changes in T. gondii (Yong et al. 1991). Platelet microsomal fractions that generate TXA2 and a TXA2 analogue (carbocyclic TXA2) also exert these toxoplasmacidal effects (Yong et al. 1991). In contrast, toxoplasmacidal activity was not exhibited by either the 13-HODE precursor linoleic acid or 12-HETE. These studies suggest that the surface membranes of T. gondii are susceptible to disruption by selected linoleic acid and arachidonic acid derivatives. Our findings suggest that the following sequence of events could occur when platelets interact with T. gondii. Release of TXA2 and 13-HODE by the T. gow