Clin. exp. Immunol. (1976) 26, 375-380.

Specific antibody-dependent killing of Toxoplasma gondii by normal macrophages S. E. ANDERSON, JR.*, S. C. BAUTISTA & J. S. REMINGTON Division of Allergy, Immunology and Infectious Diseases, Palo Alto AMedical Research Foundation and Department of Medicine, Division of Infectious Diseases Stanford University School of Medicine, Palo Alto, California, U.S.A. (Received 1 June 1976)


The requirement for specificity of antibody-dependent inhibition or killing of intracellular Toxoplasma gondii trophozoites by normal mouse peritoneal macrophages was evaluated in vitro using light microscopy and autoradiography. Anti-toxoplasma antibody in the presence of 'accessory factor' rendered extracellular T. gondii trophozoites non-viable and non-infectious for cells, whereas exposure of extracellular trophozoites to heat-inactivated immune serum did not appear to damage the parasites. Although pretreatment of extracellular trophozoites with heatinactivated immune serum neither diminished nor prevented infection of normal mouse peritoneal macrophages, it did confer upon macrophages the ability to inhibit or kill the organisms once they were intracellular. In contrast, pretreatment of trophozoites with either heat-inactivated normal or Besnoitiajellisoni immune serum did not enable normal macrophages to inhibit or kill T. gondii; rather, such organisms multiplied intracellularly in normal macrophages. Thus, pretreatment with specific antibody alone prepared T. gondii trophozoites for intracellular destruction by normal mouse peritoneal macrophages. These results suggest that specific antibody acting in concert with normal macrophages may play a role in controlling infection with T. gondii.

INTRODUCTION Infection with the obligate intracellular protozoan, Toxoplasma gondii, results in the development of both humoral and cellular immune responses (Remington & Krahenbuhl, 1976). In vivo and in vitro studies, in both experimental animal models (Frenkel, 1967; Remington, Krahenbuhl & Mendenhall, 1972; Strannegard & Lycke, 1972; Vischer & Suter, 1954) and man (Anderson & Remington, 1974; Borges & Johnson, 1975; Ruskin & Remington, 1976) have demonstrated that cell-mediated immunity (CMI) is paramount among host defence mechanisms against T. gondii. From these studies, it appears that the macrophage is the effector cell of this CMI. Nevertheless, there is substantial evidence suggesting that antibody is also important in controlling or preventing infection due to T. gondii (Krahenbuhl, Ruskin & Remington, 1972; Lycke et al., 1965; Vischer & Suter, 1954). Previous studies from our laboratory demonstrated that normal human monocyte-derived macrophages, cultured in vitro, were readily infected by T. gondii trophozoites and were incapable of inhibiting replication of or killing the parasites, but rather supported their intracellular multiplication. However, incubating T. gondii trophozoites with heat-inactivated homologous immune serum, prior to infection, enabled non-immune human monocyte-derived macrophages to inhibit or kill the parasites when intracellular (Anderson & * Special Fellow, Bureau of Medicine and Surgery, U.S. Navy. Present address: Division of Infectious Diseases, Department of Medicine, National Naval Medical Center, Bethesda, Maryland, U.S.A. Correspondence: Dr. J. S. Remington, Palo Alto Medical Research Foundation, 860 Bryant Street, Palo Alto, California

94301, U.S.A.



S. E. Anderson, Jr., S. C. Bautista S J. S. Remington

Remington, 1974). Similarly, non-immune, non-activated mouse peritoneal macrophages support the intracellular multiplication of T. gondii, but are able to inhibit or kill trophozoites which have been pretreated with heat-inactivated immune serum (Jones, Len & Hirsch, 1975; Sethi et al., 1975; Stadtsbaeder, Nguyen & Calvin-Preval, 1975). This study was designed to investigate in vitro whether specificity of the antibody is required for such interaction between macrophages and trophozoites.

MATERIALS AND METHODS Infiction of experimental animals. Chronic infection with Toxoplasma gondii and Besnoitia jellisoni was established as previously described in random-bred female ICR/SIM or retired breeder Swiss Webster mice (Ruskin, McIntosh & Remington, 1969). Collection oj immune sera. One month after infection, mice were bled from the axillary artery and sera from each group of infected mice were pooled and stored at -20'C. The serum pools obtained will be referred to as Toxoplasma (TIS) or Besnoitia (BIS) immune serum. Control serum (CS) from age, sex, and weight-matched normal uninfected mice was prepared and stored in the same manner. Antibody determination. The Sabin-Feldman dye test (DT) (Frenkel & Jacobs, 1958) and the indirect fluorescent antibody (IFA) test (Remington, Miller & Brownlee, 1968) for Toxoplasma antibody determinations were performed as previously described and the titres expressed as the initial dilution of the serum. The fluorescein-conjugated antiserum to mouse IgG was obtained from Meloy Labs, Incorporated, Springfield, Virginia, Lot No. FGM201. When tested in the DT, TIS and BIS had titres of 1:4096 and 1: 512 respectively, whereas CS had a titre of < 1:4 indicating a lack of measurable Toxoplasma neutralizing antibody. TIS had a titre of 1:1000 and BIS had a titre of 1:100 in the appropriate IFA test; CS was negative ( 0 2).

inoculation experiments (100% of mice died within 6 days following i.p. inoculation of 1 x 106 organisms). Thus, these differences were due to an inhibition of [3H]UdR uptake in TIS-treated T. gondii and the dissolution of such organisms within the macrophages during the early hours of infection. DISCUSSION Anti-toxoplasma antibody in the presence of 'accessory factor' (the complement-properdin system) (Feldman, 1956; Sabin & Feldman, 1948) rapidly damages extracellular T. gondii trophozoites resulting in non-viable organisms as determined by inability of the parasites to incorporate [3H]UdR (Anderson & Remington, 1974), rendering them nonstainable with methylene blue (Sabin & Feldman, 1948), collapse and partial lysis of the cell membrane with extrusion of cytoplasmic contents (Klainer, Krahenbuhl & Remington, 1973; Lelong & Desmonts, 1952; StrannegArd, 1967), and neutralizing their infectivity for animals and cells in tissue culture (Anderson & Remington, 1974; Jacobs et al., 1959; Lycke et al., 1965). In contrast, as shown in this study and by others, trophozoites exposed to TIS which has been heat-inactivated to remove 'accessory factor' show no evidence of damage by either light or electron microscopy (Jones et al., 1975; Strannegard, 1967), are capable of incorporating [3H]UdR (Anderson & Remington, 1974), exclude trypan blue dye (Sethi et al., 1975), and are infective for mice (Anderson & Remington, 1974; Sabin & Feldman, 1948). Although such pre-infection treatment with heat-inactivated TIS neither diminished nor prevented infection of normal macrophages, it did empower the macrophages to inhibit or kill the organisms once they were intracellular. This finding confirms and extends the results of other in vitro studies using normal macrophages from mice (Jones et al., 1975; Sethi et al., Stadtsbaeder et al., 1975). Similar results have also been reported using normal macrophages from guinea-pigs (Vischer & Suter, 1954), and humans (Anderson & Remington, 1974). Lycke et al. (1965) have suggested that this antibody, which neutralizes the infectivity of T. gondii for cells, is distinct from the 'cytoplasm modifying' antibody demonstrated in the DT since the latter requires the presence of 'accessory factor' to be active.

Antibody-dependent killing of T. gondii


This effect of antibody-treatment on the uptake of [3H]UdR by intracellular T. gondii was quantitatively evident within the first hour of infection, suggesting that inhibition or killing of the organisms occurred very early after their entry into the macrophages. Such rapid inhibition or killing of intracellular T. gondii has also been demonstrated in activated mouse peritoneal macrophages (Remington et al., 1972), although pretreatment of the organisms with antibody was not required. That killing of the parasites, rather than mere inhibition of RNA synthesis and multiplication of the organisms, occurred in the vast majority of TIS-treated T. gondii was supported by the microscopic evidence of fragmented remnants of the organisms within cytoplasmic vacuoles (Anderson & Remington, 1974). A few organisms in the TIStreated challenge inoculum which infected macrophages were normal in staining and morphologic characteristics, took up [3H]UdR, and multiplied intracellularly at the expected generation time (i.e., 4-6 hr [Jones et al., 1975]). Thus, it appears that the method used in this study for pretreatment of T. gondii with antibody did not adequately 'prepare' all the organisms for killing by macrophages (Anderson & Remington, 1974). A less likely explanation would be the presence of a heterogenous population of infected macrophages (i.e. a subpopulation of macrophages incapable of inhibiting or killing T. gondii). In order to demonstrate this effect of antibody alone on T. gondii, extracellular trophozoites must be exposed to the heat-inactivated TIS prior to infecting the macrophages. Once T. gondii are intracellular, addition to the medium of antibody with or without 'accessory factor' has no demonstrable effect on the interaction of the organisms with the macrophage (Anderson & Remington, 1974; Jones et al., 1975; Sabin & Feldman, 1948). Matsubayashi & Akao (1966) demonstrated by electron microscopy that while ferritin-conjugated anti-toxoplasma antibody was able to infiltrate into macrophages, it was unable to come into contact with the organisms. To determine the specificity of the antibody required for enabling normal macrophages to destroy T. gondii, we also challenged the macrophages with heat-inactivated CS and BIS-treated organisms. Besnoitiajellisoni is a protozoan which is closely related to T. gondii; morphologically similar and sharing some common antigens (probably cytoplasmic and not cell wall) demonstrable by immunodiffusion and haemagglutination techniques (Lunde & Jacobs, 1965). The lack of cross-reaction between B. jellisoni and T. gondii in the DT (Hoff& Frenkel, 1974; Lunde & Jacobs, 1965) or IFA test (Goldman, Carver & Sulzer, 1957), which demonstrate antibody to cell-wall antigens, was confirmed in the present study. Despite the close taxonomic relationship and common antigens of B. jellisoni and T. gondii, heatinactivated BIS-treatment of T. gondii failed to enable macrophages to destroy the organisms. Thus, antibody specific for the infecting organism appears to be required to bestow upon normal macrophages the ability to kill T. gondii. The mechanism by which specific antibody 'prepares' T. gondii for destruction intracellularly was not elucidated but Jones et al., (1975) have provided electron microscopic evidence that the presence of antibody stimulates the fusion of lysosomes with phagosomes containing T. gondii, resulting in degeneration of the organisms. In vitro investigations of the effect of antibody-treatment, prior to infection of macrophages, of a variety ofother micro-organisms against which CMI is important in host defense, have led to a divergence of findings. For example, antibody pretreatment of Leishmania parasites (Mauel et al., 1975) and Mycobacterium tuberculosis (Armstrong & Hart, 1975), did not result in destruction of these organisms by macrophages from normal guinea-pigs or from normal mice, respectively. However, antibody pretreatment of other intracellular parasites such as Besnoitia jellisoni (Hoff & Frenkel, 1974), Brucella melitensis (Ralston & Elberg, 1969), Rickettsia mooseri (Gambrill & Wisseman, 1973), and vaccinia virus (Silverstein, 1970) resulted in inhibition or destruction of the micro-organisms by macrophages. Specific antibody in the presence of 'accessory factor' apparently contributes to host defence mechanisms by its lethal effect on extracellular T. gondii, while CMI is the primary defense against organisms in their intracellular habitat which are sheltered from the effects of circulating antibody. In addition, the results of this study support a role in host defence against T. gondii for specific antibody acting in concert with non-immune macrophages. This may be especially important in controlling the early acute phase of infection prior to the establishment of effective CMI.


S. E. Anderson, Jr., S. C. Bautista & J. S. Remington

This work was supported by grant AI-04717 from the National Institutes of Health. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. REFERENCES ANDERSON, S.E., JR & REMINGTON, J.S. (1974) Effect of microscopic studies on Toxoplasma gondii. Amer. 1. trop. normal and activated human macrophages on Toxoplasma med. Hyg. 15, 486. gondii. 3. exp. Med. 139, 1154. MAUEL, J., BEHIN, R., BIRouM-NOERJAsIN & BODIL HOLLE, ARMSTRONG, J.A. & HART, P. D'ARcY (1975) Phagosome(1975) Studies on protective cell-mediated mechanisms in lysosome interactions in cultured macrophages infected experimental Leishmania infections. Mononuclear with virulent tubercle bacilli. 3. exp. Med. 142, 1. Phagocytes in Immunity, Infection and Pathology. (ed. BORGES, J.S. & JOHNSON, W.D., JR (1975) Inhibition of by R. Van Furth), p. 663. 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Specific antibody-dependent killing of Toxoplasma gondii by normal macrophages.

The requirement for specificity of antibody-dependent inhibition or killing of intracellular Toxoplasma gondii trophozoites by normal mouse peritoneal...
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