Jpn. J. Protozool. Vol. 35, No. 1. (2002)

3

Review

Host immune system against Toxoplasma infection
Hideyuki NAGASAWA
National Research Center for Protozoan Diseases, Obihiro University of Agriculture
and Veterinary Medicine, Obihiro 080-8555, Japan.

Toxoplasma gondii is an intracellular protozoan parasite belonging to the subclass Coccidia.
Toxoplasmosis is widespread in human beings andmany other warm-blooded animals. The cats andother felines are the definitive hosts known to harbor the sexual stage of this parasite. Although
Toxoplasma is an umcommon cause of disease inindividuals with a normal immune system, immunocompromised hosts such as AIDS patients areat high risk of developing severe toxoplasmosis,
especially toxoplasmic encephalitis as opportunistic infections (Sibley and Boothroyd, 1992).
Cell mediated immunity plays a crucial role inprotective immune responses against Toxoplasma
infection (Sethi et al., 1975; Nagasawa, 1984).
However, the protective mechanisms involved inresisting infection with a strain of low virulence(Beverley strain) of T. gondii differ greatly fromthose involved in resisting infections with a highly
virulent strain (RH strain) by the following results.
When mice were immunized with homogenate of
Toxoplasma before infection with a lethal dose of
Beverley strain bradyzoites, the mice acquired resistance and survived. By contrast, vaccination witha sublethal dose of live Beverley strain bradyzoites
was required for acquisition of resistance to infection with the highly virulent RH strain (Nagasawa
et al., 1991). These findings seem consistent withobservations that immunization with Toxoplasma
homogenate along with adjuvant failed to prevent
infection of mice by tachyzoites of the RH strain.

Heat shock protein
Exposure of cells to a variety of stressful conditions such as infection, immunization, elevatedtemperature, or stressful chemical intoxication leadto the transcription of a highly conserved set ofgenes and, subsequently, to the synthesis of a family of polypeptides called heat shock proteins(HSPs) (Lindquist, 1986; Schlesinger, 1986; Pelham, 1988). Among the various HSPs, a 65-kDa
mycobacterial HSP has been identified as a target of
T cells, including γδ T cells (van Eden et al., 1988;
Res et al., 1988; Koga et al., 1989; O’Brien et al.,
1989; Holoshitz et al., 1989; Haregewin et al.,1989). This HSP contains a highly conserved sequence and cross-reactivity with antigens frommany of other microbes. T cells reactive to HSP65
derived from pathogen exhibit cytotoxicity tomacrophages expressing host-derived HSP65(Koga et al. 1989), suggesting that HSP contributes
to the elimination of intracellular parasites as target
antigens on the infected host cells. Moreover,
HSP65 is known as one of the ligands of γδ T cells(O'Brien et al., 1991), which participate in protection against early phase of infections with intracellular pathogens (Hiromatsu et al., 1992; Raziuddin
et al., 1992).
Relationship between the expression of
HSP65 and protective immunity
When mice were immunized with Toxoplasma
homogenate, HSP65 was detectable in peritoneal

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Jpn. J. Protozool. Vol. 35, No. 1. (2002)

macrophages from these mice after immunization
but was not detectable from either unimmunized
controls or Toxoplasma homogenate themselves.
Furthermore, mice that acquired resistance against
a high-virulence RH strain after the resolution of
infection with Beverley strain bradyzoites strongly
expressed HSP65 in their peritoneal macrophages.
Moreover, a subset of γδ T cells has been shown torecognize HSP65 (O’Brien et al., 1989; Holoshitz
et al., 1989; Haregewoin et al., 1989) and was foundto increase rapidly in peripheral blood of patientswith acute T. gondii infection (De Paoli et al. 1992;
Scalise et al. 1992). Thus, it is not surprising that γδ
T cells recognizing HSP65 and involved in protective immunity in some kinds of infection including
toxoplasmosis. This T cell subset is thought possibly to represent a first line of defense against infection and is probably demonstrable in normal
individuals. Furthermore, these HSP65-reactive γδ
T cells should have been primed previously bycontact with many different microbes or by exposure to HSP generated in host cells under various
stressful conditions. We showed earlier that treatment of BALB/c mice with anti-Thy1.2 mAb one
day before immunization with Toxoplasma homogenate led to an almost complete loss of theexpression of HSP65. To determine the subsets of T
cells responsible for induction of this protein, mice
were depleted of γδ T cells, αβ T cells, CD4 + T cells
or CD8 + T cells by treating with corresponding
mAbs before immunization. From these experiments, γδ T cells were shown to be essential for the
expression of HSP65, although CD4 + αβ T cells
also contributed to some extent (Nagasawa et al.,1994).
Neospora caninum
Neospora caninum is an intracellular protozoan parasite closely related to T. gondii (Dubey etal., 1988). It is frequently diagnosed as the cause ofbovine abortion of epidemic proportion worldwide(Dubey and Lindsay, 1996). Vertical transmission

contributes significantly to the spread of N. caninum, with most congenital infections resulting in
the birth of healthy calves (Bjorkman et al., 1996;
Pare et al., 1996; Schares et al., 1998).
Fas and Fas ligand
Fas and FasL interaction is closely associatedwith immune privilege and probably provides abarrier to prevent pathogens from damaging tissues
in privileged sites. Moreover, the expression of Fas
was up-regulated by IFN-γ on human peripheral
blood T cells in vitro (Oyaizu et al., 1994). Numerous microbial pathogens have been reported to either induce or prevent apoptosis of their mammalian host cells (Zychlinsky and ansonetti, 1997). It
was reported that T. gondii infection caused apoptosis in Peyer’s patch T cells (Liesenfeld et al.,1997) and eyes (Hu et al., 1999) of mice. This
apoptosis was Fas-dependent and mediated by IFNγ. However, several papers have shown that T.
gondii infection seems to protect cells from death
induced by a number of agents (Goebel et al., 1998;
1999; Nash et al., 1998). Therefore, several contrasting views regarding the modulation of apoptosis following T. gondii infection exist.
We have earlier shown that N. caninum causes
apoptosis in IFN-γ-treated BALB/3T3 clone A31
fibroblasts in vitro (Nishikawa et al., 2001). In thelight of the close relationship between T. gondii andN. caninum, and the opposing reports on themodulation of apoptosis following T. gondii infection, in the present study we sought to investigate
the mechanism(s) of the prevention/induction of
apoptosis in IFN-γ-induced host cells infected with
T. gondii, and N. caninum parasites in an in vitro
system.
Apoptosis in host cells infected with T. gondii
or N. caninum
The data we have generated from viability
assay, FCM and TUNEL analyses, and DNA ladderobservation clearly indicated substantial apoptosis

Jpn. J. Protozool. Vol. 35, No. 1. (2002)

5

FasL

IFN-γ
Fas

cytoplasm
caspase-8
Toxoplasma
nucleuscaspase-3
DNA fragmentation
Fig. 1. Apoptosis signals in Toxoplasma-infected cells.

in IFN-γ treated and N. caninum-infected cells
compared with T. gondi-infected cells. Killed Neospora parasites failed to induce apoptosis, implyingthat parasite invasion of host cells is essential in the
induction of apoptosis by IFN-γ.
The significant activation of caspase-3 and -8,
and the inhibition of apoptosis in the presence of
caspase-3 and –8 inhibitors in N. caninum-infected
and IFN-γ treated cells compared to T. gondiiinfected cells, and the untreated and uninfected
control group, suggest the crucial role of these proteins in the induction of apoptosis in Neosporaparasitized cells. In the light of earlier reports ofmitochondrial releases of caspase-9 during apoptotic process (Marsh et al., 1995; Krajewski et al.,
1999; Susin et al., 1999), it is highly likely that both
caspase-9 and Bcl-2 play a supportive role in the
induction of apoptosis in host cells infected with N.
caninum. On the other hand, the resistance of T.
gondii-infected cells to apoptosis may be attributed

to prevention of caspase-3 and –8 activation (Fig.
1).
Similar levels of Fas-expression in cells infected with either T. gondii or N. caninum, and thepost-administration of anti-Fas mAb revealed theinduction of apoptosis in cells infected with either
species, showing insignificant differences in infection rate in apoptotic cells. The Fas-mediatedapoptosis in T. gondii-infected cells corroborates
earlier findings of an IFN-γ induced Fas-dependent
apoptosis in T cells in the Payer’s patches in
C57BL/6 mice following peroral infection with T.
gondii (Liesenfeld et al., 1997). We have noted aswell significantly higher FasL expression in N.
caninum-infected cells compared to T. gondiiinfected cells, and the decrease in cell viability inthe presence of anti-FasL mAb was dosedependent.
In the presence of IFN-γ, the number of T.
gondii tachyzoites increased in host cells, but not in

6Jpn. J. Protozool. Vol. 35, No. 1. (2002)
Toxoplasma

NO, O2-

cytokines
αβγδγδ : γδ T cellΜφαβ : αβ T cell
activation of macrophageactivation of T cells

ΜφΜφ : macrophage

NO, O2Μφcytokinesγδ

: Toxoplasma antigen

αβγδToxoplasma
specific T cellΜφ

NO, O2Μφ: host HSP

αβtoxoplasmacidal effect by
antigen specific killer T cell

HSP65 specific T cellFig. 2. Role of HSP65 for protective immunity against infection with Toxoplasma gondii.
N. caninum. It may be inferred then that the inhibition of apoptosis seems to prolong the life of theinfected-host cells and promote the multiplicationof T. gondii in the host. Also, the inhibition of
apoptosis in T. gondii-infected cells may be interpreted to suggest increased susceptibility to theparasite.
Conclusion
Characterization of effective and regulatory
functions of HSPs and apoptosis in other hosts andmicrobial systems should provide insight intomechanisms of virulence and protective adaptations
that control virulence. Thus, it seems likely that
HSPs and apoptosis could assume a critical importance in numerous host-parasite relationships, including resistance of host to otherwise destructively
virulent parasites.
From these bases, the biological role of HSP65
and apoptosis may be as follows. As first step after
infection with a low-virulence of Toxoplasma, circulating γδ T cells recognize either Toxoplasma-

derived HSP65 or host-derived HSP65, and thenthey accumulate and activated. At the second step,
macrophages activated by γδ T cells probably via
certain cytokine pathways exhibit enhanced respiratory burst releasing noxious molecules, e.g. oxygen metabolites, a major protective mechanism
against intracellular pathogens like T. gondii. As
the third step, activated macrophages synthesize
endogenous HSP65 for protection against these
toxic molecules, for repairment of damaged functions of themselves or for effective antigenpresentation. Finaly, either γδ T cells and αβ T cells
reactive to HSP65 or αβ T cells specific for
Toxoplasma antigen further accumulate and are
activated. Such T cells directly destroy the hostmacrophages or activate macrophages to kill the
intracellular Toxoplasma parasites (Fig. 2). Thishypothesis is considered by the role of host cellderived HSP. However, it has been reported thathighly virulent strain of Toxoplasma expressed highlevel of HSP compared with low virulent strain.
Because a function of HSP is to rescue the cellJpn. J. Protozool. Vol. 35, No. 1. (2002)

death, HSP derived from highly virulent strain of
Toxoplasma has a function to suppress apoptosis
which are effective to eliminate intracellular parasites in host cells. Moreover, it is of interest to investigate the relationship between HSP and apoptosis in protective immunity against Toxoplasma
infection.
REFERENCES
Bjorkman, C., Johansson, O., Stenlund, S., Holmdahl, O.J. and Uggla, A. (1996) Neospora
species infection in a herd of dairy cattle. J.
Am. Vet. Med. Assoc., 208, 1441-1444.
De Paoli, P., Basaglia, G., Gennari, D., Crovatto,
M., Modolo, M.L. and Santini, G. (1992)
Phenotypic profile and functional characteristics of human gamma and delta T cells
during acute toxoplasmosis. J. Clin. Microbiol., 30, 729-731.
Dubey, J.E., Carpenter, J.L., Speer, A., Topper, M.
J. and Uggla, A. (1988) Newly recognizedfatal protozoan disease of dogs. J. Am. Vet.
Med. Assoc., 198, 1269-85.
Dubey, J.P. and Lindsay, D.S. (1996). A review of
Neospora caninum and neosporosis. Vet.
Parasitol., 67, 1-59.
Goebel, S., Luder, C.G. and Gross, U. (1999) Invasion by Toxoplasma gondii protects humanderived HL-60 cells from actinomycin Dinduced apoptosis. Med Microbiol Immunol., 187, 221-226.
Goebel, S., Luder, C.G., Lugert, R., Bohne, W. and
Gross, U. (1998) Toxoplasma gondii inhibits the in vitro induced apoptosis of HL-60
cells. Tokai J Exp Clin Med., 23, 351-356.
Haregewoin, A., Soman, G., Hom, R.C. and Finberg, F.W. (1989) Human γδ+ T cells respond to mycobacterial heat-shock protein.
Nature (London), 340, 309-312.
Hiromatsu, K., Yoshikai, Y., Matsuzaki, G., Ohga,
S., Muramori, K., Matsumoto, K., Bluestones, J.A. and Nomoto, K. (1992) A pro-7

tective role of γ/δ T cells in primary infection with Listeria monocytogens in mice. J.
Exp. Med., 175, 49-56.
Holoshiz, J., Koning, F., Cologanm, J.E., Bruyn, J.
D. and Strober, S. (1989) Isolation of CD4 CD8 - mycobacteria-reactive T lymphocytes
clones from rheumatoid arthritis synovial
fluid. Nature (London), 339, 226-229.
Hu, H.S., Schwartzman, J.D., Yeaman, G.R,,
Collins, J., Seguin, R., Khan, I.A. andKasper, L.H. (1999) Fas-FasL interaction
involved in pathogenesis of Ocular
toxoplasmosis in mice. Infect. Immun., 67,
928-935.
Koga, T., Wand-Wurttenberger, A., Debryun, J.,
Munk, M.E., Schoel, B. and Kaufmann, S.H.
E. (1989) T cells against bacterial heat shockprotein recognized stressed macrophages.
Science, 245, 1112-1115.
Krajewski, S., Krajewska, M., Ellerby, L.M.,
Welsh, K., Xie, Z., Deveraux, Q,L.,
Salvesen, G.S., Bredesen, D.E., Rosenthal,R.E., Fiskum, G. and Reed, J.C. (1999) Release of caspase-9 from mitochondria during
neuronal apoptosis and cerebral ischemia.
Proc. Natl. Acad. Sci. U S A, 96, 5752-5757.
Liesenfeld, O., Kosek, J.C. and Suzuki, Y. (1997)
Gamma interferon induces Fas-dependent
apoptosis of Payer’s patch T cells in micefollowing
peroralinfection
withToxoplasma gondii. Infect. Immun., 65,4682-4689.
Lindquist, S. (1986) The heat-shock responses.
Ann. Rev. Biochem., 55, 1151-1191.
Marsh, A.E., Barr, B.C., Sverlow, K., Ho, M.,
Dubey, J.P. and Conrad, P.A. (1995) Sequence analysis and comparison of ribosomal DNA from bovine Neospora to similar coccidial parasites. J. Parasitol., 81, 530535.
Nagasawa, H. (1984) Immune responses in experimental toxoplasmosis in mice treated with

8

Jpn. J. Protozool. Vol. 35, No. 1. (2002)

carrageenan. Jpn. J. Parasitol., 33, 265-273.
Nagasawa, H., Manabe, T., Maekawa, Y., Oka, M.
Himeno, K. (1991) Role of L3T4+ and Lyt2+ T cell subsets in protective immune responses of mice against infection with a low
and high virulent strain of Toxoplasma
gondii. Microbiol. Immunol., 35, 215-222.
Nagasawa, H., Hisaeda, H., Fujioka, H., Aikawa,
M. Himeno, K. (1994) γδ T cells play a crucial role in the expression of 65 000 heatshock protein in mice immunized with
Toxoplasma antigen. Immunology, 83, 347352.
Nash, P.B., Purner, M.B., Leon, R.P., Clarke, P.,
Duke, R.C. and Curiel, T.J. (1998) T.
gondii-infected cells are resistant to multiple
inducers of apoptosis. J. immunol., 160,
1824-1830.
Nishikawa, Y., Mishima, M., Nagasawa, H., Igarashi, I., Fujisaki, K., Otsuka, H. and Mikami,
T. (2001) Interferon-gamma-induced apoptosis in host cells infected with Neospora
caninum. Parasitology, 123, 25-31.
O’Brien, R.L., Happ, M.P., Dalla, A., Palmer, E.,
Kubo, R. and Born, W.K. (1989) Stimulation of a major subset of lymphocytes expressing T cell receptor γδ by an antigen
derived from Mycobacterium tuberculosis.
Cell, 57, 667-674.
O'Brien, R.L.; Happ, M.P.; Dallas, A., Cranfill, R.,
Hall, L., Lang, L., Fu, Y.X., Kubo, R. andBorn, W. (1991) Recognition of a single
hsp-60 epitope by an entire subset of γδ Tlymphocytes. Immunol. Reviews, 121, 155170.
Oyaizu, N., McCloskey, T.W., Than, S., Hu, R.,
Kalyanaraman, V.S. and Pahwa, S. (1994)
Cross-linking of CD4 molecules upregulates
Fas antigen expression in limphocytes by
inducing interferon-γ and tumor necrosis
factor-α secretion. Blood, 84, 2622-2631.
Pare, J., Thurmond, M.C. and Hietala, S.K. (1996)

Congenital Neospora caninum infection indairy cattle and associated calfhood mortality. Can. J. Vet. Res., 60, 133-139.
Pelham, H. (1988) Heat shock proteins: Coming infrom the cold. Nature (London), 332, 776777.
Raziuddin, S., Telmasani, A.W., El-Awad, M.E.H.,
Al-Amari, O. and Al-Janadi, M. (1992) γδ T
cells and the immune response in visceralleishmaniasis. Eur. J. Immunol., 22, 11431148.
Res, P.C.M., Scharr, C.G., Breedveld, F.C., VanEden, W., VanEmden, J.D., Cohen, I.R. and
De Vries, R.R.P. (1988) Synovial fluid T
cell reactivity against 65 kd heat shock protein of mycobacteria in early chronic arthritis. Lancet, 11, 478-480.
Scalise, F., Gerli, R., Castellucci, G., Spinozzi, F.,
Fabietti, G.M., Crupi, S., Sensi, L., Britta,
R., Vaccaro, R. and Bertotto, A. (1992)
Lymphocytes bearing the γδ T-cell receptor
in acute toxoplasmosis. Immunology, 76,
668-670.
Schares, G., Peters, M., Wurm, R., Barwald, A. andConraths, F.J. (1998) The efficiency of vertical transmission of Neospora caninum indairy cattle analysed by serological techniques. Vet. Parasitol., 80, 87-98.
Schlesinger, M.J. (1986) Heat shock proteins: The
research for functions. J. Cell. Biol., 103,
321-325.
Sethi, K.K., Pelster, B., Suzuki, N., Piekarski, G.
and Brandis, H. (1975) Immunity to
Toxoplasma gondii induced in vitro in nonimmune mouse macrophages with specifically immune lymphocytes. J. Immunol.,
115, 1151-1158.
Sibley, L.D. and Boothroyd, J.C. (1992) Virulent
strain of Toxoplasma gondii comprise asingle clonal lineage. Nature (London), 359,
82-85.
Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo,Jpn. J. Protozool. Vol. 35, No. 1. (2002)

I., Brenner, C., Larochette, N., Prevost, M.
C., Alzari, P.M. (1999) Kroemer, G. Mitochondrial release of caspase-2 and -9 during
the apoptotic process. J. Exp. Med., 189,
381-394.
Van Eden, W., Thole, J.E.R., Van der Zee, R.,
Noordzij, A., Can Emden, J.D., Hensen, E.J.
and Cohen, I.R. (1988) Cloning of the mycobacterial epitope recognized by Tlymphocytes in adjuvant arthritis. Nature(London), 331, 171-173.
Zychlinsky, A. and Sansonetti, P.J. (1997) Apoptosis as a proinflammatory event: what can
we learn from bacteria-induced cell death?
Trends Microbiol., 5, 201-204.

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