Toxoplasma inhibitory factor and interleukin 2 : assessment of their effects on toxoplasma gondii
replication within a continuous macrophage-like cell line
by Anita Susan Hagemo
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Veterinary Science
Montana State University
© Copyright by Anita Susan Hagemo (1982)
Abstract:
A rapid, reproducible, and accurate microassay was developed to study the effects of soluble
lymphocyte products on Toxoplasma replication with in parasitized cells. The assay system is based
upon radioactive uracil incorporation into nucleic acids of intracellular T. gondii and employs a
homogeneous macrophage-like cell line, RAW 264. Supernatant fluids prepared from antigen- and Iect
in-stimulated murine spleen cells effectively inhibited Toxoplasma replication within RAW 264 cells
and induced proliferation of the interleukin 2 (IL2)-responsive T-cell line, CTLL2C175. These results
indicated that both Toxoplasma inhibitory factor (TIF) and IL2 were present in unfractionated
supernatant fluids. Removal of IL2 activity from antigen- and lectin-prepared supernatant fluids after
adsorption by CTLL 2C17 5 cells had little effect on inhibiting Toxoplasma replication within RAW
264 cells. Supernatant fluids prepared from concanavalin A-stimulated FS6-14.13 cells, an
IL2-producing T-cell hybridoma, induced proliferation of CTLL 2C17 5 cells, but failed to inhibit T.
gondii replication within RAW 264 cells. Fractionation of lectin-prepared supernatant fluids by
gel-filtration using Sephadex G-100 column chromatography showed that TIF activity eluted in a broad
peak in the region of 39,000 to 58,000 daltons, whereas IL2 activity eluted in the molecular size range
of 28,000 to 30,000 daltons. Therefore, both TIF and IL2 were found to be present in the same antigenand lectin-prepared supernatant fluids and experimental results suggest that these soluble lymphocyte
products are two distinct and unique molecules. TOXOPLASMA INHIBITORY FACTOR AND INTERLEUKIN 2:
ASSESSMENT
OF THEIR EFFECTS ON TOXOPLASMA GONDII REPLICATION WITHIN
A CONTINUOUS MACROPHAGE-LIKE CELL LINE
by '
Anita Susan Hagemo
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Veterinary Science
MONTANA STATE UNIVERSITY
Bozeman, Montana
December 1982
N3
H J9 0 ^
Cop.9"
APPROVAL
of a thesis submitted by
Anita Susan Hagemo
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
/ ^ 4 ^
_________
Chairperson, Graduate Committee
Date
Approved for the Major Department
V
Date
y-_
Approved for the College of Graduate Studies
Date
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of
the requirements for a master's degree at Montana State
University, I agree that the Library shall make it
available to borrowers under rules of the Library.
Brief
quotations from this thesis are allowable without special
permission,
provided that accurate acknowledgment of
source is made.
Permission for extensive quotation from or reproduc­
tion of this thesis may be granted by my major professor,
or in his absence, by the Director of Libraries when, in
the opinion of either, the proposed use of the material is
for scholarly purposes.
Any copying or use of the material
in this thesis for financial gain shall not be allowed
without my written permission.
V
ACKNOWLEDGMENTS
Dr. Paul Baker, the author's major advisor,
is greatly
appreciated for his enthusiasm, guidance, and continuing
support throughout the graduate program.
The author wishes
to thank Dr. Glen E p ling, her co-major advisor,
understanding,
for his
encouragement, and advice, and Dr. J . R .
Dubey, her minor advisor,
area of Parasitology.
for guidance and training in the
Many thanks are extended to the
author's graduate committee members for their aid and help­
ful suggestions in the preparation of this manuscript.
Sincere gratitude is expressed to Carolyn Gay for
typing the manuscript with speed and excellence and to
Merrie Mendenhall for professional photographic services.
Thanks are extended to Ken Knoblock for his time and
patience in training the author laboratory techniques.
The
author is very grateful to Ken Knoblock and Bill Tino for
technical assistance, advice, and friendship, and to Doris
Do, a fellow graduate student and close friend,
for her
continuing encouragement in the completion of this degree.
The author is indebted to her fiance, Richard
Vickstrom, for his. unyielding support and patience during
the graduate program, and to her parents, Margaret and Roy
Hagemo, for their encouragement and confidence in her
abilities and decisions.
I
I
vi
TABLE OF CONTENTS
Page
VITA.... ........
iv
ACKNOWLEDGMENTS.................
LIST OF TABLES ..................... ........... .
v
viii
LIST OF FIGURES................. ........ .......... .
ix
ABSTRACT..... ..........
xi
CHAPTER
1
INTRODUCTION............................
I
2
LITERATURE REVIEW.......................
6
3
MATERIALS AND METHODS... ............
Animals......................
Medium....... .....................
Toxoplasma Strains.......................
Cells..............
Immunization, ..........
Toxoplasma Lysate Antigen..........
Preparation of Antigen- and Lectin-Stimulated
Lymphocyte .Supernatant Fluids............
Assessment of TIF Activity ........ .......... .
Assay for IL2 Activity...........
Adsorption of IL2 Activity from TIF-Containing
Supernatant Fluids......
Ammonium Sulfate Precipitation...........
Fractionation Procedure..........
III Vivo Evaluation of TIF-Containing Super­
natant Fluids......... ...............
4
RESULTS... ......................
Generation of Immune Splenic Supernatant Fluid
with Optimum TIF Activity ...................
Antitoxoplasma Activity in Normal Peritoneal
Macrophages Incubated with Lectin-Induced
TIF-Containing Supernatant Fluid...........
26
26
26
28
29
31
32
32
34
36
37
37
38
39
40
40
40
Vii
CHAPTER
/
Page
Microscopic Observation of Restricted
Toxoplasma Replication in Normal Peritoneal
Macrophages Pretreated with TIF-Containing
Supernatant Fluids...... .................. .
Generation of Antitoxoplasma Activity in
Tachyzoite-Infected RAW 264 Cells Pretreated
with TIF-Co ntain in g Supernatant Flu idsi....„
Microscopic Observation of Restricted %. gondii
Replication in RAW 264. Cells Pretreated
with Immune TIF-Containing Supernatant
F Iu id ..... ........... .................. .
IL2 Activity in Antigen- and Lectin-Stimulated
Lymphocyte Supernatant Fluids.............
Pretreatment of Tachyzoite-Infected RAW 264
Cells with Supernatant Fluid Prepared from
Lectin-Stimulated FS6-14.13 Cells........
Removal of IL2 Activity from TIF-Containing
Supernatant Fluids After Adsorption with
CTLL2C175 Cells. ..............................
Reduced Antitoxoplasma Activity in RAW 264
Cells Pretreated with Adsorbed TIF-Containing
Supernatant Fluids....
.............
Gel-Filtration.... ............................. .
Treatment of Mice with TIF-Containing Super­
natant Fluids......
41
49
54.
57
58
59
62
66
68
5
DISCUSSION......................................
70
6
SUMMARY............
88
REFERENCES CITED
91
viii
LIST OF TABLES
Table
Page
1.
Antitoxoplasma activity in normal peritoneal
macrophages pretreated with antigen-stimulated
splenocyte.supernatant fluids..............41
2.
Intracellular replication of _T. gondii within
normal peritoneal macrophages pretreated with
TIF-containing supernatant fluids.......... . .... 43
. 3.
Intracellular replication of T_. gondii within
RAW 264 cells pretreated with immune TIFcontaining supernatant fluid..........'.......... 54
4.
IL2 activity in antigen- and lectin-stimulated
splenocyte supernatant fluids.................... 57
5.
IL2 activity in FS6-derived supernatant
fluids........ ........ ....... ................... 58
6.
IL2 activity in immune TIF-containing super­
natant fluids after adsorption with
CTLL2C175 cells___ _____ ■......................... 61
7.
IL2 activity in lectin-induced TIF-containing
supernatant fluids after adsorption with.
CTLL 2 0175
cells. .. ......
62
Effect of TIF-containing supernatant fluids on
Toxoplasma infactivity in mice inoculated with
gondii oocysts..............
69
8.
LIST OF FIGURES
Page
F igure
1.
2.
Inhibition of [ H]-uracil incorporation by
T_. qondii tachyzoites in normal peritoneal
macrophages pretreated with Iectin-indue ed
mouse TIF-containing supernatant fluid....
Normal macrophages pretreated with immune and
lectin-in due ed splenic supernatant fluids
and infected with tachyzoites.
A-F,
Light micrographs of preparations fixed
16 h after addition of culture medium.
A.
immune TIF control: unrestricted multi­
plication of organisms within cytoplasmic
vacuoles......
44
B.
immune T I F : rounding up of only two
organisms within a single cytoplasmic
vacuole. ..................................... ...45
C.
lectin-induced mouse T I F : presence of
two organisms within a single cytoplasmic
vacuole........
46
lectin-induced mouse TIF control: rosette
of organisms within a single cytoplasmic
vacuole..........
..47
lectin-in due ed rat TI F : restricted multi­
plication of organisms within a single
cytoplasmic vacuole........
47
D.
E.
F.
3.
....42
lectin-induced rat TIF control: unrestricted
multiplication of organisms within cyto­
plasmic vacuoles..... ........ ........... . ... 483
Inhibition of [ H ]-uracil incorporation by
T_. gondii tachyzoites in RAW. 264 cells
pretreated with immune TIF-containing
supernatant fluid................ ....... .
5I
X
LIST OF FIGURES (continued)
F igur e
4.
Page
Inhibition of [ ]-urac il incorporation by
T. gondii tachyzoites in RAW 264 cells
pretreated with lectin-induced mouse TIFcontaining supernatant fluid........... ...... ..
5.
Inhibition of ["3
*
5Hj-Uracil incorporation by
_T. gondii tachyzoites in RAW 264 cells
pretreated with lectin-induced rat TIFcontaining supernatant fluid....... .....53
6.
RAW 264 cells pretreated with immune splenic
supernatant fluid and infected with
tachyzoites.
A - B , Light micrographs of.
preparations fixed 16 h after addition of
culture medium.
7.
8.
9.
10.
A.
immune TIF control: rounding up of several
organisms within a single cytoplasmic
vacuole............. ........ ........ ...55
B.
immune T I F : restricted multiplication of
organisms within a single cytoplasmic
vacuole.............. .................... .
52
56
3
Incorporation of [ H ]- urac il by _T. gondii
tachyzoites in RAW 264 cells pretreated with
FS6-deriv ed supernatant fIuids................... 60
3
Incorporation of [ H ]-uracil by _T. gondii
tachyzoites.in RAW 264 cells pretreated with
adsorbed and unadsorbed immune TIF-containing
supernatant fluids..................... .......... 64
3
Incorporation of [ H ]-urac il by T_. gondii
tachyzoites in RAW 264 cells pretreated with
adsorbed and unadsorbed lectin-in due ed rat
TIF-containing supernatant fluids...........
65
IL2 activity and TIF activity assayed after
Sephadex G-100 column chromatography of
lectin-stimulated rat splenic supernatant
fluid............. ................................ 6.7
ABSTRACT
A rapid, reproducible, and accurate microassay was
developed to study the effects of soluble lymphocyte
products on Toxoplasma replication with in parasitized
cells.
The assay system is based upon radioactive uracil
incorporation into nucleic acids of intracellular _T. gondii
and employs a homogeneous macrophage-like cell line, RAW
264.
Supernatant fluids prepared from antigen- and Iect instimulated murine spleen cells effectively inhibited
T oxoplasma replication within RAW 264 cells and induced
proliferation of the interleukin 2 (IL2)-responsive T-cell
line* CTLL2C115.
These results indicated that both
T dxoplasma inhibitory factor (TIF) and IL2 were present
in unfractionated supernatant fluids.
Removal of IL2
activity from antigen- and Iectin-prepared supernatant
fluids after adsorption by CTLL 2C1 75 cells had little
effect on inhibiting Toxoplasma replication within RAW 264
cells.
Supernatant fluids prepared from concanavalin
A-stimulated FS6-14.13 cells, an IL2-producing T-cell
hybridoma, induced proliferation of CTLL 2C1 75 cells, but
failed to inhibit Tv gondii replication within RAW 264
cells.
Fractionation of lectin-prepared supernatant
fluids by gel-filtration using Sephadex G-10Q. column
chromatography showed that TIF activity eluted in a broad
peak in the region of 39,000 to 58,000 daltons, whereas
IL2 activity eluted in the molecular size range of
28,000 to 30,000 daltons.
Therefore, both TIF and IL2
were found to be present in the same antigen- and lectinprepared supernatant fluids and experimental results
suggest that these soluble lymphocyte products are two
distinct and unique molecules.
I
CHAPTER I
INTRODUCTION
Toxoplasma gondii is a coccidian protozoan capable of
infecting a wide range of vertebrate cells (2,30,31).
■Al­
though the mechanism of entry of this obligate intracellular
parasite into host cells was demonstrated to occur by phago­
cytosis (52,80) , the predominant means of cellular entry
was recently established to occur by active invasion (80).
Intracellular tachyzoites reside in cytoplasmic vacuoles
and replicate asexually with a generation time of five
to ten hours (52), leading eventually to cell rupture and
liberation of parasites, which then infect adjacent
cells.
Even though the cells are eg uipped with extensive
biochemical machinery including Golgi apparatus, ribosomes,
and endoplasmic reticulum (40), extracellular replication
of Ti. gondii was never reported.
Although macrophages are highly phagocytic scavenger
cells that kill and degrade many microbes, Ti. gondii was
reported to survive and multiply within these mammalian
cells (53).
After phagocytosis of this intracellular
parasite by macrophages,
it was demonstrated that living
1[. gondii alter the phagosomal membrane such that fusion
with lysosomes does not occur (52).
In contrast, dead
2
Toxoplasma or Toxoplasma coated with antibody are readily
engulfed and degranulat ed by lysosomal constituents after
phagocytosis by macrophages
(52).
The mechanism(s ) of resistance to
completely understood.
qondii is not
Humoral immune reactions are of
diagnostic importance in infections with Toxoplasma, but
the antibody titer does not necessarily signify immune
status against the agent (27)..
It was shown that antibody
inactivates or lyses Toxoplasma in the presence of a com­
plement-like "accessory factor" obtained from Toxoplasma
antibody-free human serum, which is the basis of the SabinFeldman dye-test (93).
Antibody is effective, however,
only against free or extracellular organisms, whereas the
intracellular organisms are protected.
Since most of the
life cycle takes place intracellularly, humoral immunity
does not play a major role in protection.
Several investigators (12,28,48,54,87) reported that
cell-mediated immunity is important in resistance to in­
fection with this obligate intracellular pathogen.
Lymphoid
cells adoptively transferred from Toxoplasma-immune donors
confer specific immunity to Toxoplasma infection in hamsters
(28).
In addition, activated macrophages are important
effectors of immune protection to infection with %. gondii
(74,87).
Activated macrophages from mice chronically
3
infected with T_. gondii had an enhanced capacity for killing
the parasite when compared with macrophages from normal
mice (87).
Shirahata and coworkers (101) observed that nonimmune
peritoneal mouse macrophages cultivated iji vitro with super­
natant fluids prepared from lymphocytes of Toxoplasma-immune
mice stimulated with Toxoplasma lysate antigen significantly
inhibit the intracellular replication of T^. gondii.
In
contrast, a marked proliferation of organisms was noted
in the same cells incubated with supernatant fluids ob­
tained from immune lymphocytes cultured without antigen.
It was later shown that this antitoxoplasma activity
observed is due to a soluble lymphocyte product,
T oxoplasma growth inhibitory factor (Toxo-GIF)
T oxoplasma inhibitory factor (TIF)
prepared supernatant fluids.
called
(102) or
(53), present in antigen-
This lymphokine, with a re­
ported molecular weight of 38,000 to 55,000 daltons (79,
103), can also be generated by stimulating lymphocytes of
nonimmune animals with the T-cell lectin, concanavalin A
(Con A) (102).
In addition to sharing roughly similar molecular
weights, the production of TIF occurs under conditions
similar to those required for production of another
T-cell derived product previously known as T-cell
growth factor (TCGF)
(35), and currently referred
4
to as interleukin
2 (IL2)
(I).
For example, TIF was re­
ported to be elaborated by j^n vitro reactivation of lympho­
cytes from dye-test negative, streptokinase-stre ptodornas e
(SK-SD)
positive individuals with SK-SD, or from lymphocytes
of dye-test positive individuals with Toxoplasma lysate
antigen (5).
Similarly,
production of IL2 was demonstrated
to result from i_n vitro reactivation of lymphocytes from
ovalbumin-immune animals with ovalbumin
(56).
Moreover,
both TIF and IL2 were reported to be generated by stimu­
lating murine spleen cells with mitogenic doses of Con A
(35,102).
Thus, in the present study it was hypothesized that
TI F , a lymphokine which inhibits the intracellular repli­
cation of T . gondii within parasitized phagocytic cells
(101), might be the same as IL2, a lymphokine which was
previously reported to only activate other T-cells (35).
To determine whether TIF and IL2 were actually one and
the same molecule, a rapid, reproducible, and accurate
microassay using a homogeneous macrophage-like cell line,
RAW 264, was developed to study the effects of these
soluble lymphocyte products oh Toxoplasma replication
within parasitized cells.
This microassay, which was
based upon the observation by Pfefferkorn and Pfefferkorn
(85) and Schwartzman and Pfefferkorn (96) that radioactive
uracil is not significantly incorporated into nucleic acids
of host cells, but is incorporated into nucleic acids of
5
actively replicating tachyzoites, has proven to be a useful
method for determining the relationship among H F
and IL2.
Experimental results indicated that (I) both TIF and IL2
were present in the same antigen- and lectin-prepared
supernatant fluids; and (2) these soluble lymphocyte
products are in fact two distinctly separate molecules.
t
J
6
CHAPTER 2
LITERATURE REVIEW
Toxoplasma gondii, an obligate intracellular coccidian
protozoan, is a common cause of infection and disease in
many vertebrates including man (2,31).
Serological
studies indicate that about 40% of the United States pop­
ulation has had toxoplasmosis by age 30 (31).
Although
toxoplasmosis is commonly asymptomatic, it. can produce
serious illness in congenitally infected children (18,25,
69) and in immunosup pressed patients (14,32,81).
In
veterinary medicine, it is a significant cause of abortions
in sheep (10) and is therefore of economic importance.
iT-
The life cycle of T . gondii is similar to most coccidia
because it has an e nt er oepith elial cycle in a specific
host, resulting in the formation of oocysts (29).
Only
domestic cats and certain other members of the family .
Felidae, the definitive hosts for Ti. gondii, were reported
to produce Toxoplasma oocysts (70).
Unlike most other coccidia, jT. qo.ndii has an extraintestinal or tissue cycle'.
These stages, which also occur
in cats, appear to constitute the entire life cycle in
non felines (31).
In acute visceral infections, rapidly
7
dividing Toxoplasma (tachyzoites) multiply in any cell of
1
the intermediate host (non felines) and in nonintestinal
epithelial cells of the definitive host (21).
Cysts con­
taining s lowly multiplying organisms (bra dyzoites) are
characteristic of chronic infections and occur in the
brain, heart, skeletal muscle, and other tissues (31).
The three known infective stages of T^. gondii are
bradyzoites (in cysts), tachyzoites (in pseudocysts), and
sporozoites (in oocysts)
(21).
It was reported that the
three modes of Toxoplasma transmission, carnivorism,
fecal
contamination, and transplacental or congenital infection,
are linked to the life cycle (29).
These modes of trans­
mission involve the different stages as follows:
ingestion
of bradyzoites, tachyzoites, or both; contamination with
feline feces containing sporozoites of spofulated oocysts;
or transplacental infection of the fetus with tachyzoites
after ingestion of encysted bradyzoites or sporulated
oocysts by the mother (21).
T_. gondii is capable of infecting a wide range of
vertebrate cells (30).
Intracellular tachyzoites reside
in cytoplasmic vacuoles and replicate asexually with
a generation time of five to ten hours (52)', leading
eventually to cell rupture and liberation of parasites,
which then infect adjacent cells.
Even though the cells
are equipped with extensive biochemical machinery including
8
mitochondria, Golgi apparatus, ribosomes, and endoplasmic
reticulum (40),
extracellular replication of
gondii
was never reported.
Although the mechanism of entry Of1JT. gondii into
host cells was demonstrated to occur by phagocytosis (52,
80), the predominant means of cellular entry was recently
established to occur by active invasion (80).
When
examined ultrastructurally, it was clear that different
events occur during these two entry processes (80).
Nichols
and O'Connor (80) reported that microfilament aggregates
are present beneath attached organisms during phagocytosis,
whereas such aggregates are absent from phagocytic cells
during invasion.
The absence of subplasmalemmaI filament
aggregates and disruption of host cell membrane during
invasion indicated that cellular penetration is due to
effects exerted by the parasite (80).
Although lacking
definitive evidence, membrane disruption seen during in­
vasion is speculated to be due to the release of lytic
enzymes from specialized organelles in J[. gondii called
rhoptries (82).
Another feature distinguishing phagocytic from in­
vasive entry is the presence of two distinct types of
parasite-containing vacuoles in Toxoplasma infected cells.
During phagocytosis of JT. gondii by host cells, the para­
sites are immediately enclosed in typical phagocytic
vacuoles (80).
In contrast, as parasites disrupt the host
9
cell plasma membrane during invasion, they are not
immediately enclosed in intact vacuoles.
Instead, large
parasitophorous vacuoles quickly form around the parasites
which invade (80).
Vacuoles formed after invasion
have tubules protruding into their cavities, whereas
phagocytic vacuoles have less free space around the
parasites and absence of tubules (80).
Since during
invasion there is no evidence of an in vaginat ed host
cell plasma lemma external to the unit membrane of the
parasite (80), the formation of a. classical phagocytic
vacuole does not occur during this entry process.
Although macrophages are highly phagocytic scavenger
cells that kill and degrade many microbes,
T_. gondii was
reported to survive and multiply within these mammalian
cells (53).
After phagocytosis of this intracellular
parasite by macrophages, it was demonstrated that living
2» gondii alter the phagosomal membrane such that fusion
with lysosomes does not occur (52).
In contrast,
dead
Toxoplasma or Toxoplasma coated with antibody are readily
engulfed and degranulated by lysosomal constituents after,
phagocytosis by macrophages (52).
The mechanism (s ) by which living J_. qond ii can survive
within phagocytic cells remains largely unknown.
Since
lysosomal constituents are not delivered to phagocytic
vacuoles harboring living organisms, T_* Qondii is specu­
lated to secrete some substance that alters the vacuolar
10
membrane and prevents lysosomal fusion (53).
Jones and
Hirsch (53) suggested that the fate of intracellular
Toxoplasma may reflect cellular factors in which there are
variations in the capacity of macrophages to destroy this ■
pathogen.
However, when highly activated macrophages con­
taining large numbers of lysosomal granules were compared
with unstimulated macrophages containing fewer granules,
no differences were observed in the capacity of these cells
to kill ingested organisms
(53).
Jones and Hirsch (53) observed that early after phago­
cytosis and throughout the course of parasite multiplication
in the cell,
phagocytic vacuoles harboring living, organisms
are enveloped by endoplasmic reticulum and mitochondria.
The mechanism accounting for this phenomenon is unknown,
but these investigators suggested that attraction for
these cytoplasmic organelles might reflect an alteration
in the vacuolar membrane.
Another morphologic feature indicating an alteration
in the vacuolar membrane by T_. gondii is the presence of
microvillus protrusions of the membrane into the vacuole
forming arrays of small ,vermiform structures (100).
Since
these microvillus protrusions markedly increase the surface
area of the vacuolar membrane,
Jones and Hirsch (53) sug­
gested that they may serve a role in transferring to the
phagocytic vacuole some cytoplasmic factors required for
parasite survival or multiplication.
11
Several different mechanisms might be responsible
for parasite survival in phagocytic cells, but morphologic
evidence indicates that vacuolar membrane alteration by
T_. gondii may be of importance in relation to the absence
of lysosomal fusion (53).
Such membrane alteration may also
play a role in protecting the host cell or in nourishing
the parasite.
The mechanism(s)
of resistance to
completely understood.
. gondii is not
Humoral immune reactions are of
diagnostic importance in infections with Tv gondii, but the
antibody titer does not necessarily signify immune status
against the agent (27).
It was reported that antibody
inactivates or lyses Toxoplasma in the presence of a
complement-like "accessory factor" obtained from Toxoplasma
antibody-free human serum, which is the basis of the
Sabin-Feldman dye-test (93).
Antibody is effective only
against free or extracellular organisms, whereas the
intracellular parasites are protected.
Studies on potential immune mechanisms associated with
resistance to Tv gondii showed that at least two types of
humoral antibodies,
complement-fixing and cytoplasm-modify­
ing, are elicited by animals inoculated with live parasites.
(114).
Subseguent immunity to reinfection was reported to
develop in these animals (114).
However, only one type of
humoral antibody, the cytoplasm-modifying antibody of Sabin
and Feldman (93), was found to be elicited by animals
12
inoculated with heat-killed Ioxoplasma antigen, and no
immunity to reinfection develops (58).
Cutchins and Warren
(15) reported that a .cytoplasm­
modifying antibody titer and protection against reinfection
are demonstrated in guinea pigs inoculated with a killed
vaccine.
They also found that inoculation of these animals
with a killed vaccine, to which an adjuvant had been added,
results in the production of a higher cytoplasm-modifying
antibody titer and active protection against reinfection,
as well as a substantial complement-fixing antibody titer.
According to the report by Foster and McCulloch (27),
neither vaccination nor recovery from infection completely
prevents the persistence of Tv gondii in various organs of
guinea pigs.
These investigators showed that even in the
presence, of very high antibody levels, it is still possible
to isolate living Toxoplasma from the tissues of these
animals.
It was therefore suggested that immunity to toxo­
plasmosis in guinea pigs and probably man involves a
restriction or repression of growth of the parasites,
rather than complete destruction (27).
Others later showed that immunization of mice with a
few cysts of a low virulent strain of Tv gondii elicits both
a humoral and cellular immunity which are manifested in
a resistance to intraperitoneal challenge with a virulent
strain of the parasite (54,98). ■ Antibody titers,
detectable
by the Sabin-Feldman dye-test, are then evident throughout
the life of the animal.
Cell-mediated immunity is important in resistance to
infection caused by intracellular pathogens (12,28) .
Lymphoid cells adoptively transferred from Toxoplasma-immune
donors confer specific immunity to Toxoplasma infection in
hamsters (28).
In addition, activated macrophages are
important effectors of immune protection to infection with
T_. gondii (74,87) .
Activated macrophages from mice chroni­
cally infected with %. gondii had an enhanced capacity for
killing this parasite when compared with macrophages from
normal mice (87).
The mechanism whereby these cells
acquire their enhanced ability to inhibit intracellular
replication or kill this pathogen is still unclear.
A number of investigators (4,12,47,48,vS4,59,98,101)
reported that macrophages cocultivated with sensitized
lymphocytes and mitogen or antigen, to which the lymphocytes
were previously sensitized, acquire the ability to kill or
inhibit replication of the obligate intracellular parasite,
T_. qon dii.
Jones and coworkers (54) demonstrated that
peritoneal macrophages obtained from Toxoplasma infected
animals are unable to kill or inhibit the intracellular
replication of T^. gondii.
Reex po sure of these cells in
vitro to lymphocytes of Toxoplasma-immune mice and
specific antigen
(54) or supernatant fluids obtained
from, immune lymphocytes stimulated with Toxoplasma
14
lysate antigen
(12) restores the ability of immune macro­
phages to inhibit the organism.
Exposure of normal
macrophages to the same lymphocytes and antigen or super­
natant fluids does not result in macrophage activation
since no antitoxoplasma activity is observed in these cells
(12,54). Thus, for induction of immunity, these investi­
gators reported that macrophages must be in a state of
"stimulate" before they are exposed to such antigenstimulated immune lymphocytes or their products.
Shirahata et al.
(101) showed that when glycogen-
induced peritoneal macrophages from non immune mice are
cultured in_ vitro with supernatant fluids prepared from
lymphocytes of Toxoplasma-hyperimmunized mice stimulated
with Toxoplasma lysate antigen, significant inhibition of
intracellular replication of J_. gondii results.
In con­
trast, a marked proliferation of organisms was noted in
the same cells incubated with supernatant fluids obtained
from immune lymphocytes cultured without antigen (101).
These investigators reported that stimulation of immune
lymphocytes with specific antigen is necessary for the pro­
duction of biologically active products capable of confer­
ring cultured nonimmune macrophages the ability to kill
JT. gondii or inhibit its intracellular replication (101).
According to the report by Sethi and coworkers (97),
about 8 0?o of the macrophage population obtained from thioglycollate-stimulated nonimmune mice could kill and digest
15
intracellular !_• gondii when cocultivated with Toxoplasmaimmune lymphocytes and Toxoplasma lysate antigen.
They also
noted that only 20% of the macro phage population obtained
from, unstimulated nonimmune mice were able to destroy intra­
cellular organisms, although the cells were cultured in
the presence of the same lymphocytes and antigen.
In human cases, Borges and Johnson
(12) reported that
supernatant fluids obtained from antigen-stimulated
lymphocytes of T oxoplasma-immune subjects induce macro­
phages from immune and nonimmune subjects to inhibit intra­
cellular replication of T ox op lasma.
They also showed that
these macrophage populations cocultivated with T ox oplasmaimmune lymphocytes and specific antigen inhibit parasite
replication as effectively as antigen-stimulated immune
lymphocyte supernatant fluids.
Anderson et al.
(5) also demonstrated that human
macrophages incubated with supernatant fluids prepared
from antigen-stimulated lymphocytes of individuals who
were positive in the Sabin-Feldman dye-test acquire the
ability to inhibit intracellular _T. gondii replication.
Supernatant fluids obtained from no ns ens itized lymphocytes
of Sabin-Feldman dye-test negative subjects and T ox oplasma
lysate antigen are ineffective.
However, incubation of
macrophages with supernatant fluids prepared from lympho­
cytes of dye-test negative, streptokinase-streptodornase
(SK-SD) positive individuals when cultured in the presence
16
of SK-SD or concanavalin A (Con A) were found to induce
a significant, but lesser activation (5).
According to the report by McLeod and Remington (66),
a variety of iri vivo and _in vitro conditions initiates
or modulates stimulation of macrophages by different
mechanisms, leading to specific or nonspecific effects.
These investigators showed that immunization of mice
with Corynebacterium
parvum, a stimulus unrelated to
Besnoitia j el I is oni and JT. gondii, results in the ability
of the host's macro phages to nonspecif ically. kill both
protozoa.
Nonspecific microbicidal processes of macro­
phages were also observed in cells stimulated with a variety
of agents and substances including infection with intra­
cellular microorganisms
(9,26,34,49,61,115), endotoxin
(3), RNA (6), and soluble lymphocyte products elaborated
from antigen- or mitogen-stimulated lymphocytes (4).
Bio logically active moieties,
antibody,
present in the supernatant fluid of antigen-
or lectin-stimulated lymphocytes,
(88).
other than soluble
are termed " Iy m phok in es "
These mediators of cellular immunity are soluble
products .of lymphocytes and are responsible for the
multiple effects
of a cellular immune reaction.
Regarding
lymphokines in relation to Toxoplasma infection, Shirahata
and coworkers (101) reported that in response to antigenic
stimulation, spleen cells from Toxoplasma-immune mice
elaborate a soluble factor which is capable of inhibiting
17
the intracellular replication of T_. gondii within immune
and nonimmune peritoneal mouse macrophages.
This soluble
factor, termed Toxoplasma growth inhibitory factor (ToxbGIF)
(102)
or simply Toxoplasma inhibitory factor (TIF)
(55), can also be generated by stimulating spleen cells
from nonimmune animals with the T-cell lectin, Con A.
( 102 ) .
In order to determine the molecular characteristics
of T l F , supernatant fluids prepared from lectin- and
antigen-stimulated murine spleen cells were fractionated
by gel-filtration using Sephadex G-100 column chroma­
tography and assayed for TIF activity (79,103).
Factor
activity was reported to elute from Sephadex G-100 in
the molecular size range of 38,000 to 55,000 da!tons (79,
103).
When factor activity in these column fractions was
further examined after purification by isoelectric
focusing (IFF,
pH 4.0 - 7.0 gradient),
TIF activity was
found to fractionate with an isoelectric
point of pH 4.9
to 5.9 (79,103).
Chinchilla and Frenkel (13) reported that jm vitro
reactivation of lymphocytes from Toxplasma-immune hamsters
with Tox plasma lysate antigen results in the production
of a soluble factor which is capable of inhibiting the
intracellular replication of T^. gondii within immune and
nonimmune peritoneal hamster macrophages.
In contrast
to murine T I F , this soluble factor, with a reported
18
molecular weight of 4,000 to 5,000 daltons (13), is also
effective against Toxoplasma infection in hamster kidney
cell and fibroblast cultures.
Antitoxoplasma activity in macrophages exposed to;
lymphocytes of Toxoplasma-immune mice and specific antigen
or supernatant fluids obtained from these antigen-stimu­
lated lymphocytes was previously assessed by direct micro­
scopic observation of parasite multiplication or inhibition
of multiplication
(98,101,102).
Since visual evaluation of
Gi emsa-stained preparations was both time-consuming and
subject to potential observer bias, McLeod and Remington
(65,67) developed a new method for evaluating the capacity
of phagocytic cells to kill or inhibit multiplication of
intracellular X* gondii.
This new method was based on the observation by
Pfefferkorn and Pfefferkorn (85) and Schwartzman and
Pfefferkorn (96) that radioactive uracil is not signifi)
cantly incorporated into nucleic acids of host cells, but
is incorporated into nucleic acids of actively replicating
Toxoplasma tachyzoites.
These investigators reported that
cells infected with X« gondii incorporate much more uracil
into their nucleic acids than uninfected cells and that
nearly all of the label is associated with the parasites.
They ascribed the observed differences in uracil incorpor­
ation in uninfected and infected cells to differing
levels of uridine phosphorylase, an enzyme that converts.
19
uracil into uridine, which is then incorporated into nucleic
acids.
Since intracellular Toxoplasma have substantially
more uridine phosphorylase than host cells, they incor­
porate more uracil into their nucleic acids.
McLeod and Remington (65,67) subsequently reported that
when phagocytic cells are cultured with TIF-containing
supernatant fluids and are infected with JT. gondii, incor­
poration of radioactive uracil by parasites is significantly
less than identical cultures incubated with control super­
natant fluids.
They also found that uptake of radiolabel
correlates with T ox plasma multiplication or inhibition of
multiplication in G iemsa-stained preparations, and that
extracellular organisms do hot incorporate sufficient •
amounts of uracil to affect interpretation of the assay.
This technique made it possible to more accurately
assess antitoxoplasma activity in phagocytic cells acti­
vated JLn vitro by TIF-conta in ing supernatant fluids.
Since lymphokines are produced in minute quantities,
and are therefore difficult to purify, analyses of un­
purified crude supernatant fluids, which could theoretically
contain two or more active moieties, has led to confusion
in ascertaining the relationship among soluble mediators.
For example, supernatant fluids prepared from antigenstimulated splepocytes of Tox plasma-immune mice were shown
to contain a variety of biologically active substances
including TIF (55), immune interferon (IF) (104), and
20
macrophage migration inhibitory factor (MIF)
(79,103),
which were distinguished primarily by their functional
and biochemical characteristics.
In addition to sharing roughly similar molecular
weights, the production of TIF occurs under conditions
similar to those, required for production of another
T-c ell derived product
factor (TCGF)
previously known as T-c ell growth
(33), and currently referred to as inter­
leukin 2 (IL 2) (I).
F or example, TIF was reported to
be elaborated by j^n vitro reactivation of lymphocytes
from dye-test negative, streptokinase-streptodornase (SKS D ) positive individuals with SK-SD, or from lymphocytes
of dye-test positive individuals with Toxoplasma lysate
antigen
(5).
Similarly,
production of IL 2 was demonstrated
to result from iji vitro reactivation of lymphocytes from
ova lb urnin-immune animals with ovalbumin (56).
Moreover,
both TIF and IL 2 were reported to be generated by stimu­
lating murine spleen cells with Con A (35,75).
It
appears that T I F , a lymphokine which inhibits the intra­
cellular replication of T_. gondii within phagocytic cells
(101), may be similar to IL 2, a lymphokine which was pre­
viously reported to only activate other T-c ells (35).
Supernatant fluids prepared from Con A-stimulat ed
murine splenocytes were shown to contain a variety of bio­
logically active substances including T-c elI growth factor
(TCGF)
(35), costimulator (84), T-c ell replacing factor
21
(TRF)
(94), and killer helper factor (KHF)
(37).
All four
lymphokines were reported to act as communication signals
between leukocytes (I).
Gillis and Smith (35) reported that exogenously
supplied TCGF is responsible for the continuous prolifer­
ation of antigen-specific cytotoxic murine T-cells.
Paetkau et al.
(84) demonstrated that costimulator induces
mitogenes is in primary thymocyte cultures, whereas Con A
in itself is unable to stimulate these cells to proliferate.
Schimpl and Wecker (94) described a soluble factor which
is capable of completely replacing T-cells in the immune
response of B-c ells to T-c ell dependent antigens. . This
lymphokine, termed T R F , was shown to restore the Jm vitro
antibody response of murine B-c ells to sheep red blood
cells (SRBC) , a T -dependent antigen in T-c ell depleted
or in nude mouse splenocyte cultures (94).
Finally,
Gillis et a l . (37) reported that KHF is capable of in­
fluencing maturation,
proliferation, or both of prothymp-
cytes present in athymic nude mice.
They showed that
dual stimulation of nude mouse spleen cells with mitomycininactivated, allogeneic tumor cells and. KHF induces the
proliferative expansion of allo-reactive, Thy-I antigen­
positive, cytotoxic lymphocytes.
Although TCGF, costimulator, T R F , and KHF displayed
distinctive functions, several investigators (38,72,112,
113) collaborated on the purification of these four
22
lymphokines in an attempt to establish their biochemical
relationships.
Supernatant fluid prepared from Con A-
stimu,lated murine splenoc.ytes was successively fraction­
ated by gel-filtration using Sephadex G-IOO column chroma­
tography and isoelectric focusing (IE F , pH 4.0 - 7.0
gradient) and assayed for TCGF, costimulator, T R F , and
KHF activity (24,99,113).
All four activities were re­
ported to co-purify to a single molecular weight class of
3 0,000 da Iton s and separate by charge into two components
with isoelectric points of pH 4.3 and 4.9 (113).
Although
TRF was found to display an additional activity which
fractionates in the pH range of 3.0 to 4.0 (113), all four
lymphokines are considered to be the same molecule.
Thus,
based on the decision by participants at the Second Inter­
national Lymphokine Workshop (Ermatingen, Switzerland,
May 27-31, 1979), TCGF, costimulator, TRF, and KHF are
collectively referred to as interIeukin 2 (I).
In order to study the kinetics of IL 2 production and
utilization,
Gillis et a l . (36) developed a rapid microassay
for the quantitative determination of IL2 activity in super­
natant fluids prepared from I ec tin-stimulated mononuclear
cells.
Their microassay is based upon tritiated thymidine
incorporation into nucleic acids of proliferating murine
tumor-specific cytotoxic T lymphocytes
(CTLL I and CTLL 2)
(35) cultured in the presence of IL 2-co nta in in g supernatant
fluid.
The relative amount of IL 2 present in active
23
supernatant fluid can be quantitatively determined by
analyzing tritiated thymidine incorporation by CTLL cells
cultured in a standard lot of IL2, arbitrarily defined to
contain one unit/ml, compared with incorporation by cells
cultured in an unknown amount of IL2.
Gillis and coworkers
(36) reported that CTLL cells
cultured for 24 hours in the absence of exogenously
supplied IL2, yet in the presence of Con A-containing
medium, do not significantly incorporate tritiated thymidine
into their nucleic acids.
In contrast, CTLL cells cultured
in the presence of increasing concentrations of IL2-containing supernatant fluid incorporate tritiated thymidine in a
dose-dependent manner (36).
Continued proliferation
of these cells was found to progressively deplete the
amount of IL2 originally present in the lymphocyteconditioned medium (36).
This, loss of IL2 activity, which
was reported to be due to its active removal and utiliza­
tion by cytotoxic T lymphocytes, results in their diminished
proliferation and ultimate death (103).
of murine cytotoxic T lymphocyte
Long-term culture
lines, then, requires the
continous presence of exogenously supplied IL 2 , since lectin
containing medium alone is incapable of maintaining pro­
liferation.
Smith et a I .. (105) and Smith (106) reported that IL2
functions to provide the proliferative signal in the
T lymphocyte immune response. . It provides the mitogenic
24
stimulus, causing DNA synthesis, blast transformation, and
ultimately division of lymphocytes after antigen or lectin
binding to the cell membrane.
A subset of I lymphocytes
(IL2-pr oduc er cells), in cooperation with macrophages,
release IL 2 upon interaction with antigen or lectin (105).
Another subset of I lymphocytes (IL 2-responsive cells)
simultaneously respond to IL2 by undergoing blast trans­
formation and division (105).
These investigators con­
cluded from their studies that the specificity of this
proliferative response is directed by the activating signal
because only those cells that have bound antigen or lectin
become responsive.to IL2.
The requirement for lectin- or antigen-activated
macrophages in the T lymphocyte immune response was demon­
strated by several investigators (71,107).
Their studies
indicated that one mechanism by which activated macrophages
participate in the T-cell proliferative response is via
the release of a monokine called lymphocyte activating
factor (LAF) .
I (!LI)
This monokine, which was renamed interleukin
(I), is responsible for the production of IL 2 (107)
by mature, thymic-derived T lymphocytes (106).
Studies
using murine cells demonstrated that after antigen or lectin
binding and in the presence of ILl, Lyl+ amplifier T-celIs
produce IL2 (111).
Another subset of T-c elIs, Ly2,3 +
cytotoxic T-cells, which have also bound the antigen or
lectin, proliferate in response to IL2 (111).
Schreier
25
and Tees (95) suggested that Lyl+ helper T-c elIs are also
capable of responding to IL2.
Thus, there are at least
two hormone-like soluble lymphocyte products which function
to regulate the magnitude of the T-cell .proliferative
response.
26
CHAPTER 3
MATERIALS'AND METHODS
Animals
Adult female Sprague-Dawley CD rats,
purchased from
Charles River Breeding Laboratory, Wilmington, M A , were
used for obtaining spleen cells.
Female BALB/c mice,
purchased at six to eight weeks of age from the same
source as the rats, were used for obtaining peritoneal.
macrophages, spleen cells, and maintaining by serial
passage tachyzoites in peritoneal cavities and bradyzoites
in brain cysts.
Medium
Powdered RPMI 1640 medium (CAT #430-1800,
Gibco, Grand
Island, NY) was prepared to 10X concentration in distilled,
deionized water, buffered with NaHCO^, filtered through
a 0.22 urn filter, and stored at 4° C .
Prior to use, it
was diluted ten-fold to working concentration with sterile,
distilled,
deionized water and supplemented with 50 units/ml
penicillin (Pfizer, Groton, C T ); 50 ug/ml gentamicin
(Schering, Kenilworth, NO); 25 mM 2-mercaptoethanol (Sigma,
St. Louis, MO); and 300 ug/ml L-glutaijiine (Gibco) .
The
pH was adjusted to 7.1 with I N HCl and osmolarity adjusted
to 300 mOsm with saline or water.
27
Click's medium, purchased at 4X concentration (Altick
Associates, Hudson, WI ) was aliquoted (25 ml) into 50 ml
Falcon plastic centrifuge tubes (#2070, Falcon Plastic,
division of B ecton-Dick inson , Oxnard , CA). and stored at
-64° C .
Prior to use, it was diluted four-fold to working
concentration with sterile,
distilled,, deionized water,
buffered with NaHCO., and I M HEPES (Research Organics',
Cleveland,. OH), and supplemented with 50 units/ml
cillin (Pfizer);
ug/ml L-glutamine
peni­
50 ug/ml gentamicin (Schering); and 300
(Gibco) .
The pH was adjusted to 7.1
and osmolarity adjusted to 300 mOsm.
Iscove's Modified Dulbecco's Medium (IMDM) was prepared
from powdered Dulbecco's Modified Eagle Medium (DMEM, CAT
n
#430-2100, Gibco) dissolved to approximately 1.25X concen­
tration in distilled,
deionized water.
mented with the following:
It was then supple­
31 mM NaHCO^; 25 mM HEPES
(Research Organics); 1 20 uM cystine-HCI (Sigma); 160 mM
Na^SeO^ (Sigma);
50 uM 2-merca ptoethano I (Sigma); 14.5
uM fatty acid-free bovine serum albumin (BSA , Sigma);
1.13 mM human transferrin (Behring Diagnostics, Somerville,
Nd), 1/3-saturated with FeCl^;
131 uM asparagine
222 uM alanine
(Sigma);
(Sigma); 180 uM aspartic acid (Sigma);
410 uM glutamic acid (Sigma);
8.7 mM sodium pyruvate
(Gibco) ; 23 uM biotin (Sigma); 4.0 uM Vitamin B ^
2.0 uM glutamine
(Sigma);
(Gibco); 50 units/ml penicillin (Pfizer);
and 50 ug/ml gentamicin
(Schering).
A suspension of 19 uM
28
cholesterol
(Sigma)J 10 uM Iin oleic acid (Sigma); and 1.0
mM l-oleoyl-2-palmitoyl
phosphatidyl choline (Sigma) was
prepared in IX DMEM containing 290 uM fatty acid-free BSA
(Sigma).
This suspension was sonicated at 4° C for 10-12
min at 80 watts and added to the previous mixture at a
ratio of 1:400 (volume/volume).
The medium was brought to
working concentration with distilled,
deionized water, the
pH was adjusted to 7.1, and osmolarity adjusted to 300 mOsm.
It was then filtered through a 0.22 urn filter and stored in
500 ml aliquots at -640 C until used.
Toxoplasma Strains
The virulent RH strain of jT. gondii (92), obtained from
Dr. J . P . Dubey (Veterinary Research Laboratory, Bozeman,
MT), was maintained by serial intraperitonea I (i.p.)
passages at three to four day intervals in BALB/c mice.
Infected
mice were killed by cervical dislocation and
injected i.p. with 3.0 ml of phosphate-buffered saline (PBS)
and tachyzoites were collected from the peritoneal cavity.
Intracellular organisms were released from peritoneal macro­
phages by forcing the aspirate through a 27 gauge needle
four times. The parasites in the supernatant fluid were
sedimented by centrifugation at 350 x g for ten min and were
resuspended in the medium appropriate for the test to be
done and identified in later sections.
For establishing
chronic infection, BALB/c mice were inoculated with brain
cysts of the a virulent TC-I strain of Tv gondii (22), also
29
Obtained from Dr. J . P . Dubey.
Oocysts of the GT-I strain
of T_. gondii (22).» also obtained from Dr. J . R . Dubey, were
used to infect BALB/c mice treated with TIF-containing
supernatant fluids; one oocyst of this strain is lethal to
mice (23).
The oocysts were obtained from feline feces
as previously described (19).
C ells
The monocytic tumor cell line, RAW 264 (86), was
obtained from Dr. Paul Guyer, Dartmouth Medical School,
Hanover, N H .
F56-14.13 cells, an interleukin 2 (IL2 )-
producing T-c ell hybridoma (41), was obtained from Dr. John
Kappler, National Jewish Hospital, Denver, CO.
The cell
lines were grown at 37° C in 5% CO^ in tissue culture
flasks (#3018, Falcon Plastic,
division of B ecton-Dickins on)
and passed every three days in RPMI 1640 medium containing
20% heat-inactivated fetal calf serum (FD S , Sterile Systems,
Logan, U T ).
Splenocyte suspensions were prepared either from
unimmunized animals or from Toxoplasma-immune BALB/c mice.
Spleens were excised under aseptic conditions, rinsed in .
PBS, and placed in glass Petri dishes containing approxi- .
mately 10 ml of RPMI 1640 medium.
The spleens were minced
with small dissecting scissors and fragments teased with
sterile forceps.
The fragments were further homogenized
by repeatedly aspirating,
first through a 10 ml syringe,
30
and then through a 20 gauge needle.
The cell, suspension
was decanted into a 50 ml Falcon plastic centrifuge tube
(#2070, Falcon Plastic,
division of Becton-Dickinson) and
allowed to stand for ten min to eliminate debris.
The
splenocytes in suspension were transferred to another 50
ml plastic centrifuge tube (#2070, Falcon Plastic) and
centrifuged at 350 x g for ten min.
The pellet was resus­
pended in approximately 2 ml of RPMI 1640 medium and red
blood cells were lysed by hypoosmotic shock.
bation for ten min at room temperature,
After incu­
debris was further
eliminated by transferring the supernatant fluid to a fresh
50 ml centrifuge tube.
Following centrifugation at 350 x
g for ten min, the supernatant fluid was aspirated and
s plenocytes were resuspended in the medium appropriate for
the test to be done and identified in later sections.
Peritoneal macrophages were obtained from normal
BALB/c mice, after killing by cervical dislocation, by
washing the peritoneal cavities with 3.0 ml of PBS.
The
aspirate was centrifuged at 350 x g for ten min and the
pellet was resuspended in approximately 2 ml of PBS.
After red blood cells were lysed by hypoosmotic shock,
the cell suspension was centrifuged (350 x g, ten min)
and the pellet was resuspended in RPMI 1640 medium
containing 20% FCS..
CTLL 2C1 75 cells (8), an IL2-dependent murine T-c ell
line, were used in all IL 2 assays. . The cell line was grown
31
at 37° C in 5% CO ^ in tissue culture, flasks (#3018, Falcon
Plastic) and passed every three days in RPMI 1640 medium
containing 10% FCS and approximately I unit/ml IL2.
Immunization
Mice were immunized with bra dyzoites.
For this, BALB/c
female mice were inoculated with the avirulent TC-I strain
of 2« gondii♦
Four to eight weeks later, the mice were
killed by cervical dislocation and their brains were
examined microscopically
(400X) for cysts.
To release
bradyzoites from cysts, the brain was ground with mortar
and pestle, and incubated in 10 volumes of pepsin-HCI
solution (30)
(pepsin, Difco Laboratories, Detroit, MI)
for 60 min at 370 C .
The homogenate was washed twice with
PBS and 0.5 ml of a brain suspension was inoculated sub­
cutaneously into approximately fifty normal BALB/c female
mice.
Seven mice inoculated with peps in-digested bra dy­
zoites were killed eight weeks post-inoculation and all
had Toxoplasma cysts in their brain.
To prepare immune TIF-containing supernatant fluid,
it was necessary to determine the degree of immunity in
mice immunized with the TC-I strain of 2* gondii.
Normal
mice and mice immunized ten weeks previously (three mice
per group) were challenged with an i.p. injection of graded
doses of tachyzoites of the virulent RH strain
of 2* gondii.
Un immunized mice died with a mean survival time of only four
days when inoculated with 125 organisms, whereas immunized
mice survived ten days.
At lower challenge doses, normal
mice died with a mean survival time of ten and fifteen days
when inoculated with 60 and 30 organisms, respectively,
but all immunized mice were still alive when the experi­
ment was terminated at sixty days post-challenge.
Non­
challenge d mice, immunized at the same time as challenged
mice, were used as a source of immune splenocytes.
■Toxoplasma Lysate Antigen
Tachyzoites of the virulent RH strain of T_. gondii
7
were adjusted to a concentration of 7.8 x 10 parasites/ml
in sterile,
distilled,
deionized water, aliquoted in 1.0
ml volumes into glass vials, frozen, and lyophilized.
Prior
to use, contents of a vial were resuspended to 1.0 ml in
Click's medium containing 2% F C S .
Optimum antigen concen­
tration for preparation of immune TIF-containing supernatant
fluid was determined after preliminary experimentation.
Preparation of Antigen- and Lectin-Stimulated Lymphocyte
Supernatant Fluids
Immune TIF was prepared by culturing 10.0 ml of spleen,
cells (2.3 x 10^ cells/ml in Click's medium containing
2% FCS) from Toxoplasma-immune mice with 10.0 ml of
Toxoplasma lysate antigen (1:20, 1:40, 1:80 dilutions).
The cells were cultured in plastic tissue culture flasks
(#3012, Falcon Plastic) standing upright for 24 h at 370 C
in 3% CO 2*
Control supernatant fluid was prepared by
33
culturing spleen cells of Toxoplasma-immune mice alone For
24 h and adding Toxoplasma lysate antigen at the end of
the incubation period.
Lectin-induced TIF (mouse-derived) was prepared by
7
culturing 6.0 ml of spleen cells (1.7 x 10 cells/ml in
RPMI 1640 medium, containing 2?o FCS) from normal mice with
3 ug/ml concanavalin A (Con A , Miles Laboratories Inc.,
Elkhart, IN) for 48 h in upright tissue culture flasks
(#3012, Falcon Plastic).
Control supernatant fluid was
prepared, by culturing spleen cells of normal mice alone
for 48 h and adding an identical amount of Con A at the
end of the incubation period.
Lectin-induced TIF (rat-derived) was prepared by
culturing spleen cells (1.0 x IO^ cells/ml in IMDM) from
normal rats with 4 ug/ml Con A for 24 h in plastic flasks
(#3024, Falcon Plastic).
Control supernatant fluid was
prepared by culturing spleen cells of normal rats alone
for 24 h and adding the same amount of Con A at the end
of the incubation period.
Additionally, supernatant fluid was prepared by
culturing F56-14.13 cells (1.0 x 10^ cells/ml in IMDM) for
24 h with 4 ug/ml Con A in tissue culture flasks (#3024,
.Falcon Plastic).
Control supernatant fluid was prepared
by culturing FS6-14.13 cells alone for 24 h and adding
4 ug/ml Con A at the end of the incubation period.
34
Collected supernatant fluids were harvested by
centrifugation at. 300 x g for 20 min , filtered through a
0.45 urn filter, and refrigerated at 4° C .
Just before use,
supernatant fluids were diluted (1:2, 1:4, 1:8, 1:16) in
RPMI 1640 medium containing 20% F C S .
Assessment of TIF Activity
The ability of supernatant fluids to inhibit intra­
cellular replication of RH strain _!• Qondii tachyzoites
was determined by two different methods.
Assessment by
microscopic observation was done by plating 300 ul of cells
(either normal peritoneal macrophages or RAW 264 cells,
1.7 x IQ^ c ells/ml) in a single drop onto sterile 15 mm
round glass cover slips (Rochester Scientific Co., Inc.,
Rochester, NY) contained in sterile 35 mm plastic tissue
culture dishes (#FB-6TC, Dispo Tray, Linbro, New Haven,
C T ).
Cells were allowed to settle for 2,5 h at 370 C in
5% CO 2 and after attachment, npnadh erent cells were removed
by washing with PBS and aspirating the supernatant fluid.
Adherent cells remaining on cover slips were incubated for
an additional 19.5 h in 2.0 ml of antigen- or lectinstimulated lymphocyte supernatant fluids diluted 1:2 in
heart in fusion broth (HIB, 0.8% in RPMI 1640 medium; Difco
Laboratories).
Following incubation, the cells were washed
and inoculated for 30-90 min with 1.0 ml of Toxoplasma
6
tachyzoites (1.0 x 10 parasites/ml in RPMI 1640 medium
containing 20% FCS) .
Extracellular organisms were removed
35
by extensive washing, and cell cultures were incubated for
an additional 16 h in culture medium.
Finally, cover slips
were washed, fixed in absolute methanol, and stained with
Giemsa (American Scientific Products, R edmon, W A ) .
Macro­
phage antitoxoplasma activity was assessed by counting
the number of infected cells, both intact and ruptured,
and the number of intracellular and extracellular
organisms.
Results were recorded after counting at least
ten microscopic fields (1000X) and expressed as the mean
number of intracellular parasites per intact cell.
Star
tistical significance of results was determined by the
two independent sample test (108) according to the following equation: z = Xy - X^ / /x^. / n^ + X^ / n^.
Analysis
was based on testing for differences in mean organisms (X)
per infected cell (n) pretreated with the appropriate
control (C) or TIF-containing (T) supernatant fluids.
In order to more easily quantify the ability of TIFcontaining supernatant fluid to inhibit intracellular
replication of T_ gondii, a microassay was devised using
either normal peritoneal cells or RAW 264 cells, and
[ H ]-uracil incorporation by intracellular parasites.
Cells
(100 uI) were seeded into wells of a 96-well microtiter
plate (#76-003-05, Linbro, Subsidiary of Flow Labs, McLean,
V A ) at the concentration of 8 x 10^ cells/ml in RPMI 1640
medium containing 20% F C S .
They were then treated as in
the microscopic assay (above),
except that 200 uI of
36
individual lymphocyte supernatant fluids, diluted 1:2 in
0.8% H I B , were added to triplicate wells.
After the final
incubation period, cells were pulsed with 50 uI of
[5,6-^H]-uracil
(specific activity 48.0 C i/mmole , #NET-
368, New England Nuclear, Boston, NA) diluted to 100 uCi/ml
in RPMI 1640 medium.
After four h of additional incubation,
contents of each well were harvested with a Bellco automated
sample harvester on glass fiber filter strips (Bellco ,
Vineland, NO).
The strips were air dried and individual
well contents were counted for radioactive nucleotide in­
corporation in toluene scintillant (Liquifluor, New England
Nuclear)
counter.
using a Beckman LSlOOC liquid scintillation
Results were expressed as the mean counts per
minute (cpm) + I SEM for triplicate wells.
Assay for IL2 Activity
CTLL2C175 cells, grown in RPMI 1640 medium containing
10% FCS and approximately I unit/ml IL 2, were harvested
from log phase cultures and washed extensively in PBS.
The
cells were pelleted by centrifugation (350 x g, ten min)
and resuspended tg a concentration of 4 x 10^ cells/ml in
IMDM.
CTLL2C17 5 cells (100 u l ) were seeded into individual
wells of a 96-well flat bottom microtiter plate (#76-003-05,
Linbr o) containing 100 ul of lymphocyte supernatant fluid
serially two-fold diluted in IMDM.
bated with culture medium alone.
Control cells were incu­
Supernatant fluids were
assayed for IL 2 using a 1.0 unit/ml standard,
provided by
r
37
Dr. Kendall Smith, Dartmouth Medical School, Hanover, N H .
After 20 h incubation at 37° C in 5%, CO
cells were pulsed
3
with 50 ul of [ H ]-thymidine (specific activity 1.9 Cl/
mmole, Schwartz-Mann , Spring Valley, NY) diluted to 10
uCi/ml in IMDM.
After four h of additional incubation,
contents of each well were harvested with a Bellco auto­
mated sample harvester on glass fiber filter strips, as
described above.
Results were expressed as the c pm for
individual wells.
Adsorption of IL2 Activity from TIF-Containinq Supernatant
Fluids .
.
.
CTLL2C1/5 cells, cultured and harvested as described
above, were transferred (3 x IO^ cells) into 15 ml centri­
fuge tubes (#2095, Falcon Plastic).
The cells were pelleted
by centrifugation at 350 x g for ten min and resuspended
in splenic supernatant fluids (mouse-derived immune TIF
and rat-derived lectin-induced TIF) to 1.0 ml/tube.
Follow­
ing a six to eight h incubation period with agitation every
30 min, cells were removed by centrifugation.
Supernatant
fluids were retained and filtered through a 0.22 urn filter.
Pre-adsorption and post-adsorption supernatant fluids were
assayed for IL 2 and TIF activity.
Ammonium Sulfate Precipitation
Supernatant fluid prepared by culturing spleen cells
7
(1.0 x 10 cells/ml in IMDM) from normal rats with 4 ug/ml
Con A for 24 h was brought to a final concentration of B5?o
38
saturation with ammonium sulfate (Sigma) by gentle stirring
until dissolved at 4° C .
at 4° C for 24 to 48 h.
The solution was then maintained
The precipitated material was
collected by centrifugation at 10,000 x g for 20 min and
resuspended in approximately 5 ml of sterile,
deionized water.
distilled,
The concentrated conditioned medium was
dialyzed against 100 volumes of 0.1 M ammonium bicarbonate
buffer at 4° C and fractionated by geI-filtration using
Sephadex G-100 column chromatography.
Fractionation Procedure
Five ml of concentrated conditioned medium were layered
on a 2.5 x 85 cm Sephadex G-100 (Fine Chemicals, Inc.,
Uppsala, Sweden) column and eluted with 0.1 M ammonium bi­
carbonate buffer into 5.5 ml fractions.
The protein content
of the column fractions collected (fractions 22-39) was
monitored with the aid of an LKB Uvicord II calibrated for
absorbance at 280 nm (LKB Produkt er, Br omma, Sweden).
The
column was calibrated with the following molecular weight
(MW) standards: bovine serum albumin
ovalbumin (MW 44,000)
chrome c (MW 12,500)
(MW 69,000)
(Miles Laboratories,
(Sigma);
Inc.); and cyto­
(Miles Laboratories, Inc.).
The contents of every two 5.5 ml column fractions
collected were, then successively combined (i . e. , 22-23, 2425, etc.) and dialyzed against 1000 volumes of distilled,
deionized water at 4° C .
The dialyzed 11 ml column frac­
tions were aliquoted into 17 x 100 mm plastic tubes, frozen,
and lyophilized.
After being resuspended in 5.0 ml of
39
medium appropriate for the test to be done f the column
fractions were filtered through a 0.22 urn filter and
assayed for IL2 and TIF activity as described above,
.'
In Vivo Evaluation of TIF-Containinq Supernatant Fluids
In vivo effectiveness of immune TIF and lectin-induced
TIF was evaluated by treating mice (six mice per group)
with TIF-containing supernatant fluids.
Three days prior
to infection, and for two weeks thereafter, the groups of
mice were treated daily with 1.0 ml of splenic supernatant
fluid prepared from I) Toxoplasma-immune splenocytes
stimulated for 24 h a) with Toxoplasma lysate antigen
(immune TIF) , b) alone, with antigen added at the end
(immune TIF control) ; 2) normal rat spenocytes stimulated
for 24 h a) with 4 ug/ml Con A (lectin-induced TIF) , or
b ) alone, with Con A added at the end (lectin-induced TIF
control).
Mice treated with RPMI 1640 medium served as the
medium control group.
All reagents (inoculated i.p.) were
diluted 1:2 in 0.8% H I B .
Treated mice were inoculated
subcutaneously with 100 infective oocysts of the GT-I
strain of T_. gondii.
The number of infectious T\ gondii
in the inocula was determined by inoculating ten-fold
dilutions of oocysts into untreated mice three weeks pre­
viously (20).
Impression smears of lungs of mice that died
were examined for
gondii tachyzoites.,
40
CHAPTER 4
RESULTS
Generation of Immune Splenic Supernatant Fluid with Optimum
TIE Activity
In order to prepare immune splenic supernatant fluid
with optimum TIE activity, Toxoplasma-immune splenocytes
were incubated for 24 h with various final concentrations
(1:40, 1:80, 1:160)
of Toxoplasma lysate antigen. . Anti-
toxoplasma activity, as assessed by the microassay utilizing
3
[ H ]-uracil incorporation by Toxoplasma in normal peritoneal
macrophages, was greatest (90%) when supernatant fluid
harvested from immune s plenocytes stimulated with a final
antigen concentration of 1:40 was used (Table I).
Subse­
quent experiments were performed using immune TIF-containing
supernatant fluids prepared in this manner.
Antitoxoplasma Activity in Normal Peritoneal Macrophages
Incubated with Lectin-Induced TIE-Containinq Supernatant
Fluid
Antitoxoplasma activity in normal peritoneal macro­
phages treated with lectin-induced TIF-containing super­
natant fluid and infected with virulent RH strain tachyzoites was assessed by the microassay utilizing [^H]-uracil
incorporation by intracellular parasites.
Normal
41
macrophages cultured in the presence of lectin-induced
mouse-derived TIF diluted 1:2 in 0.8% HIB inhibited repli­
cation; incorporation of [ ] -uracil by intracellular
parasites was 1,859 + 429 cpm compared with infected,
appropriately treated control cells which incorporated
3,187 +• 614 cpm (Fig. I).
Table I.
Antitoxoplasma activity in normal peritoneal
macrophages pretreated with antigen-stimulated
splenocyte supernatant fluids.
Final Toxoplasma
antigen concen.
1:40
1:80
1:160
medium control
CPM [^H]-uracil
3,257
5,623
4,496
32,568
+
+
+
+
292
369
1,343
1,846
Percent
inhibition3
. 90
83
86
-
Percent inhibition [ Hj-uracil incorporation in infected
cultures was calculated as follows:
I-Ccpm Experimental/
cpm Control)(100%).
Microscopic Observation of Restricted Toxoplasma Replication
in Normal Peritoneal Macrophages Pretreated with TIFContaininq Supernatant Fluids
. Infected normal peritoneal macrophages pretreated with
immune or lectin-induced TIF-containing supernatant fluids
significantly ( p <0.001)
replication,
inhibited intracellular _T" Qondii
compared with the same cells pretreated with
appropriate control supernatant fluids (Table 2, Figs.! 2A- .
2F).
Normal macrophages cultured in immune TIF control
supernatant fluid and infected with _T. gondii tachyzoites
42
induced
control
Fig. I.
induced
TIF
Inhibition of [ H ]- uracil incorporation by J_.
gondii tachyzoites in normal peritonea I macro­
phages pretreated with lectin-induced mouse TIFcontaining supernatant fluid (lectin-induced TIF,
1:2).
Macrophages were cultured in the appropri­
ate control (first bar graph) or TIF-containing
(second bar graph) supernatant fluids, prior to
infection.
Bars represent SEM radionucleotide
incorporation for triplicate cultures.
43
had 6.6 (mean) parasites per cell when examined micro­
scopically (Fig. 2A).
The same cells cultured in immune
H F -containing supernatant fluid displayed a significant
reduction in the number of intracellular parasites; at
an immune TIF dilution of 1:2, only 2.5 (mean) parasites
were observed intracellularly (Fig. 2B) .
Table 2.
Intracellular replication of T_. gondii within
normal peritoneal macrophages pretreated with
TIF-containing supernatant fluids.
Intact
cells
Ruptured
cells
Immune TIF
35
4
2.5
Immune TIF control
29
7
6.6
Lectin-induced
mouse TIF
39
0
2.1
L ectin-ind uc ed
mouse TIF control
26
9 ■
5.7
L ectin-induc ed
rat TIF
42
• I
3.8
L ectin-induc ed
rat TIF control
28
11
5.8
Treatment3
Mean paras it eg,
intact cell
Cells were pretreated with the appropriate control or
TIF-containing supernatant fluids diluted 1:2 in 0.8%
H I B , prior to infection.
Total number of cells counted per ten microscopic fields
(1000X).
Supernatant fluids containing immune TIF, lectin-induced
mouse TIF or lectin-induced rat TIF significantly
(p <0.001) inhibited H gondii replication when compared
with appropriate control supernatant fluids.
44
*
F ig. 2A.
Normal macrophages pretreated with immune TIF
control supernatant fluid and infected with
tachyzoites.
Note unrestricted multiplica­
tion of organisms within cytoplasmic vacuoles.
XllOO.
Fig.
2B.
Normal macrophages pretreated with immune TIFcontaining supernatant fluid and infected
with tachyzoites.
Note rounding up of only
two organisms within a single cytoplasmic
vacuole. X 1100.
46
Similar results w ere observed when peritoneal macro­
phages were cultured in lectin-induced 11 F-co nta in in g
supernatant fluids and infected with tachyz oites.
Mouse-
derived TIF inhibited T_. gondii replication; at a
dilution of 1:2, only 2.1 (mean) parasites were observed
intrac ellularly (Fig.
2C) compared with appropriately
treated control cells which had 5.7 (mean) parasites per
infected cell (Fig.
2D).
Rat-derived TIF also inhibited
intracellular Toxoplasma replication,
although not as
effectively as immune or mouse-derived TIF.
In the presence
of rat-derived TIF diluted 1:2, tachyzoite-infected normal
macrophages had 3.8 (mean) parasites per infected cell
(Fig.
2E) compared with appropriately treated control
cells which had 5.8 (mean) parasites (Fig.
Fig.
2C.
2F).
Normal macrophages pretreated with lectin-induced
mouse TIF-containing supernatant fluid and
infected with tachyz oites. Note presence of
two organisms within a single cytoplasmic
vacuole. X 1100.
47
Fig.
2D.
Normal macrophages pretreated with lectin-induced
mouse TIF control supernatant fluid and infected
with tachyzoites. Note rosette of organisms
within a single cytoplasmic vacuole. X 1100.
Fig.
2E.
Normal macrophages pretreated with lectin-induced
rat TIF-containing supernatant fluid and infected
with tachyzoites. Note restricted multiplication
of organisms within a single cytoplasmic vacuole.
X1100.
48
9
Fig.
2F.
Normal macrophages pretreated with lectin-induced
rat TIF control supernatant fluid and infected
with tachyz oites . Note unrestricted multipli­
cation of organisms within cytoplasmic vacuoles
and liberation into extracellular environment
following lysis of infected macrophage. X 11 DO.
49
Generation of Antitoxoplasma Activity in Tachvzoite-Infected
RAW 264 Cells Pretreated with TIF-Containinq Supernatant
FIuids
In the presence of conditioned medium containing immune
TI F , tachyzoite-in feeted RAW 264 cells incorporated from
1,879 + 80 c pm (immune H F ,
HF,
1:32)
1:4) to 6,404 + 300 c pm (immune
of [^H]-uracil (Fig. 3).
supernatant fluid,
In immune H F
control
[ H]-uracil incorporation in parasitized
cells ranged from 19,471 + 2,310 c pm (immune TIF control,
1:4) to 18,845 + 1,103 c pm (immune H F
With immune H F ,
control, 1:32).
there was a strict TIF dose-dependent
3
incorporation of [ H ]-uracil into tachyzoite-infected RAW
264 cells. No dose-dependency was observed with control
supernatant fluid.
Antitoxoplasma activity in tachyzoite-infected cells
pretreated with TIF-containing supernatant fluid prepared
from lectin (Con A)-stimUlated mouse splenocytes was sub­
sequently demonstrated by the RAW 264 microassay (Fig. 4).
At a lectin-induced H F
dilution of 1:2, tachyzoite-
in fected RAW 264 cells incorporated 4,782 + 1,393 c pm of
3
[ H]-uracil, whereas appropriately treated, parasitized
control cells incorporated 22,695 + 3,776 cpm.
Addition-
ally, since non infected cells incorporated only 1,675 +
134 cpm, this experiment demonstrated that
uracil in­
corporation was dependent upon T_. gondii infection of
50
cells, as opposed to RAW 264 incorporation of radiolabel
in the absence of parasite infection.
Results obtained
from the microassay were justified since inhibition
of Toxoplasma replication was demonstrated only in
TIF-activated, tachyzoite-infected RAW 264 cells.
TIF-containing supernatant fluid prepared from lectin
(Con A )-stimulated rat splenocytes was then assayed for its
ability to inhibit Toxoplasma replication in parasitized
RAW 264 cells (Fig. 5).
In the presence of conditioned
medium containing lectin-induced rat TIF, tachyzoitein feet ed RAW 264 cells incorporated from 1,116 + 92 c pm
(lectin-induced TIF,' 1:2) to 9,678 + 841 c pm (lectininduced TI F , 1:32)
of [ ] -uracil.
control supernatant fluid,
In lectin-induced TIF
3
[ H]-uracil incorporation in
parasitized cells, ranged from 10,036 + 1,185 c pm (lectininduced TIF control, 1:2) to 12,394 + 721 c pm (lectininduced TIF control, 1:32).
The results obtained from
this microassay using lectin-induced rat TIF were similar
to those using immune TIF (above), since there was a
strict TIF dose-dependent incorporation of [ H]-uracil into
TIF treated, tachyzoite-infected RAW 264 cells.
Addition­
ally, no dose-dependency was observed with control super­
natant fluids in either case.
51
O
<
CC
ZD
m
I
vO
ltT
cr\
I
O
X
CL
O
[Toxoplasma Inhibitory Factor]
Fig. 3.
Inhibition of [ H ]-urac il incorporation by T^.
gondii tachyzoites in RAW 264 cells pretreated
with immune T IF-containing supernatant fluid.
RAW 264 cells were cultured in 1:4 - 1:32, logdilutions of immune TIF (A) or the appropriate
control ( ■ ) supernatant fluids, prior to in­
fection.
Bars represent SEM radionucleotide
incorporation for triplicate cultures.
52
induced
control
Fig. 4.
induced
TIF
infected
Inhibition of [ H ]- uracil incorporation by T_.
gondii tachyzoites in RAW 264 cells pretreated
with lectin-induced mouse TIF-containing super­
natant fluid (lectin-induced TIF, 1:2).
RAW 264
cells were cultured in the appropriate control
(first bar graph) or TIF-containing (second bar
graph) supernatant fluids, priog to infection.
Note minimal incorporation of [ H ]-uracil by
uninfected RAW 264 cells (third bar graph).
Bars represent SEM radionucleotide incorporation
for triplicate cultures.
53
=
10
[Toxoplasma Inhibitory Factor]
Fig. 5.
Inhibition of [ H ]- uracil incorporation by T_.
gondii tachyzoites in RAW 264 cells pretreated
with lectin-induced rat TIF-containing super­
natant fluid.
RAW 264 cells were cultured in
1:2 - 1:32, log dilutions of lectin-induced
TIF (A) or the appropriate control ( ■ ) super
natant fluids, prior to infection.
Bars repre
sent SEM radionucleotide incorporation for
triplicate cultures.
•54.
Microscopic Observation of Restricted T. gondii Replication
in RAW 264 Cells Pretreated with Immune TIF-Containinq
Supernatant Fluid
Infected RAW 264 cells pretreated with immune TIFcontaining supernatant fluid significantly (p <0.001)
hibited intracellular
in­
gondii replication compared with
the same cells pretreated with appropriate control superna­
tant fluid (Table 3, Figs.
Table 3.
6A-6B).
Intracellular replication of J. gondii within
RAW 264 cells pretreated with immune TIFcontaining supernatant fluid.
Intact
cells
Treatmenta
Rupt u r e d •
c ells
Mean paras it eg
intact cell
Immune TIF
77
' I
1.9
Immune TIF control
76
I
5.9
3 Cells were pretreated with immune TIF or the appropriate
control supernatant fluid diluted 1:2 in 0.8% H I B , prior
to infection,
b
C
Total number of cells counted per ten microscopic fields
(1000X).
Supernatant fluid containing immune TIF significantly
( p <0.001) inhibited Tv gondii replication when compared
with the appropriate control supernatant fluid.
RAW 264 cells cultured in immune TIF control super­
natant fluid and infected with JT* gondii tachyzoi tes had
5.9 (mean) parasites per cell when examined microscopically
(Fig. 6A).
The same cells cultured in immune TIF-c on ta inin g
55
supernatant fluid displayed a significant reduction in the
number of intracellular parasites; at an immune TIF
dilution of 1:2, only 1.9 (mean) parasites were observed
intracellularly
Fig. 6A.
(Fig. 6B).
RAW 264 cells pretreated with immune TIF control
supernatant fluid and infected with tachyzoites.
Note rounding up of several organisms within
a single cytoplasmic vacuole. X 1100.
56
Fig. 68.
RAW 264 cells pretreated with immune TIFcontaining supernatant fluid and infected with
tachyzoites.
Note restricted multiplication of
organisms within a single cytoplasmic vacuole.
XllOO.
57
IL 2 Activity in Antigen- and Lectin-Stimulated Lymphocyte
Supernatant Fluids
. Since immune TIF and lectin-induced rat TIF were pro­
duced under conditions similar to those required for pro­
ducing IL 2, each supernatant fluid was assayed for IL 2
activity
(Table 4).
Compared with a 1.0 unit/ml standard,
both T IF-containing supernatant fluids were found to contain
considerable IL 2 activity.
Supernatant fluid containing
immune TIF was calculated to contain 2.1 units/ml IL 2, while
the lectin-induced rat TIF-containing supernatant fluid was
calculated to contain 12.3 units/ml
Table 4.
(calculations not shown).
IL 2 activity in antigen- and lectinstimulated splenocyte supernatant fluids.
CPM [^H]-Tdr incorporation by CTLL 2C175
!/Dilution
Standard3
Immune
■ TIF
2
4
8
16
32
64
128
256
512
8,878
9,124
2,970
504
94
158
130
120
180
88
11,608
11,314
7,736
3,360
530
15.2
88
76
84
.38
.
CD
Lectin
TIFc
NDd
ND '
ND
ND .
12,536
6,868
1,160
126
94
74
IL 2 standard contained 1.0 unit/ml IL 2.
Immune TIF was calculated to contain 2.1 units/ml IL 2.
Lectin TIF (rat-derived) was calculated to contain 12.3
units/ml IL2.
d
ND, not done.
58
Supernatant fluid prepared from lectin (Co n A )stimulated FS6-14.13 cells was also assayed for IL 2 activity
(Table 5) and was calculated to contain 12.5 units/ml IL 2
compared with a 1.0 unit/ml standard (calculations not
shown).
The appropriate control supernatant fluid did not
contain.IL2 activity.
Table 5.
IL 2 activity in FS6-derived supernatant fluids.
CPM [3Hj-Tdr incorporation by CTLL2C175
!/Dilution
Standarda
2
4
8
16
32
64'
128
28,998
26,672
18,460
13,124
8,48 2
4,638
2,874
1,128
CD
L ectin.
Stimulated FS6
. 29,822
33,534
36,596
30,890
26,134
24,816
16,244
288
Lectin
Control FS6
1,398
1,786
190
1,478
1,120
358
848
376
3 IL 2 standard contained 1.0 unit/ml IL 2. .
k Supernatant fluid prepared from lectin-stimulated FS614.13 cells was calculated to contain 12.5 units/ml IL 2.
Pretreatment of Tachyzoite-Infected RAW 264 Cells with
Supernatant Fluid Prepared from Lectin-Stimulated FS614.13 Cells
Since FS6- derived supernatant fluid was similar to
antigen- and lectin (Con A)-stimulated splenic supernatant
59
fluids in that they all contained IL 2 activity, the
RAW 264 microassay was performed to determine whether this
supernatant fluid was also capable of inhibiting
intracellular T . gondii replication in parasitized cells
(Fig. 7).
In the presence of supernatant fluid (diluted
1:2) prepared from lectin-stimulated FS6-14.13 cells,
tachyzoite-infected RAW 264 cells incorporated 15,226 +
3
135 c pm of [ H ]-uracil, whereas appropriately treated
control cells incorporated 19,378 + 2,048 c pm.
Similar
results were obtained with 1:4 - 1:32, Iogg dilutions.
FS6-derived supernatant fluid was unable to inhibit .
I oxoplasma replication in parasitized RAW 264 cells.
Removal of IL 2 Activity from TIF-Containinq Supernatant
Fluids After Adsorption with CTLL 201/5 Cells
Immune and lectin-in due ed TIF-containing supernatant
fluids were adsorbed with C T L L 2C175 cells and then assayed
for IL2 activity.
Results of the assay indicated that
greater than 89% (0.22 unit/ml)
of the IL 2 activity was
removed from the immune TIF-containing supernatant fluid
after adsorption with CTLL2C175 cells (Table 6) compared
with unadsorbed supernatant fluid (2.1 units/ml) .
Similarly, adsorption with CTLL2C175 cells removed
greater than 98% (C 0.15 unit/ml)
of the IL 2 activity
from lectin-in due ed rat TIF-containing supernatant fluid
(Table 7) compared with unadsorbed supernatant fluid
(12.3 units/ml).
60
[Toxoplasma Inhibitory Factor]
Fig. 7.
1
Incorporation of [ H] - uracil by T^. gondii
tachyzoites in RAW 264 cells pretreated with
FS6-deriv ed supernatant fluids.
RAW 264 cells
were cultured in 1:2 - 1:32, log„ dilutions of
lectin-stimulated FS6 supernatant fluid (A) or
the appropriate control ( ■ ) supernatant fluid,
prior to infection.
Bars represent SEM radio­
nucleotide incorporation for triplicate cultures.
61
Table 6.
IL 2 activity in immune TIF-containing super-;
natant fluids after adsorption with CTLL 2C1 /5
cells.
CPM [3Hl-Tdr incorporation by CTLL2C1/5
!/Dilution
2
4
8
16
32
64
128
256
512
CD
Standar da
8,878
9,124
2,970
504
94
158
13 0
- 120
180
88
■
Una dsorbed^
immune TIF
Adsorbed
immune TIF
11,608
11,314
7,736
3,360
53 0
152
88
76
84
38
4,152
2,866
2,032
550
174
116
104
120
74
70
IL2 standard contained 1.0 unit/ml IL2.
Unadsorbed immune TIF was calculated to contain 2.1
units/ml IL2 .
Adsorbed immune TIF was calculated to contain 0.22
unit/ml IL2.
62
Table 7.
IL 2 activity in lectin-in due ed TIF-containing
supernatant fluids after adsorption with
CTLL2C175 cells.
CPM [3HJ-Tdr incorporation by CTLL2C175
!/Dilution
Standard3
2
4
8
16
32
64
128
256
512
OD
8,878
9,120
2,970
504
94
15 8
130
120
18 0
88
.
Una ds orbed.
lectin TIF
A ds orbe d
lectin TIFc
ND
ND
ND
ND
12,536
6,868
1,160
126
94
74
ND
ND
ND
ND
1,810 .
860
310
220
194
52
IL2 standard contained 1.0 unit/ml IL2.
Unadsorbed lectin TIF (rat-derived) was calculated to
contain 12.3 units/ml IL2.
Adsorbed lectin TIF (rat-derived) was calculated to
contain less than 0.15 unit/ml IL2 (exact measurement
could not be determined).
ND, not done.
Reduced Antitoxoplasma Activity in RAW 264 Cells Pretreated
with Adsorbed TIF-Containinq Supernatant Fluids
The RAW 264 microassay was performed to determine
whether adsorption with CTLL2C175 cells had an effect on
the ability of TIF-containing, supernatant fluids to inhibit
T_. qon dii replication in tachyzoite-inf ected cells.
In
63
the presence of unadsorbed immune TIF-containing super­
natant fluid diluted 1:2, tachyzoite-infected RAW 264
cells incorporated 14,169 + 544 c pm of [ H ]-uracil,
whereas cells treated with adsorbed supernatant fluid
incorporated 27,417 + 1,268 c pm (Fig. 8).
Infected cells
treated with the appropriate control supernatant fluid
incorporated 47,586 + 104 c pm indicating a 40% reduction
in the ability of adsorbed supernatant fluid to inhibit
[
]- uracil incorporation (42%) compared with unadsorbed
supernatant fluid (70%)
(calculations not shown).
In the presence of unadsorbed TIF-containing super­
natant fluid (diluted 1:2) prepared from lectinstimulated rat splenocytes, tachyzoite-infected RAW 264
cells incorporated 31,070 + 1,405 cpm of [^H]-uracil,
whereas cells treated with adsorbed supernatant fluid
incorporated 38,758 + 58 cpm (Fig. 9).
Infected cells
treated with the appropriate control supernatant fluid
incorporated 42,036 + 780 cpm indicating an approximate
70% reduction in the ability of adsorbed supernatant
3
fluid to inhibit [ H ]-uracil incorporation (8%) compared
with unadsorbed supernatant fluid (26%) (calculations
not shown) .
64
Immune Immune Adsorbed
Control
TIF
Immune
TIF
Fig. 8.
Incorporation of [ H] -uracil by T_. gondii
tachyzoites in RAW 264 cells pretreated with
adsorbed and unadsorbed immune H F -containing
supernatant fluids diluted 1:2.
Immune TIFcontaining supernatant fluid was adsorbed by
CTLL2C1/5 cells and assayed for its ability to
inhibit intracellular Toxoplasma replication.
RAW 264 cells were cultured in the appropriate
control (first bar graph), unadsorbed immune
TIF (second bar graph) or adsorbed immune TIF
(third bar graph), prior to infection.
Bars
represent SEM radionucleotide incorporation
for triplicate cultures.
65
Lectininduced
control
Lectininduced
HF
Adsorbed
le ctin induced TIF
3
Fig. 9.
Incorporation of [ H]-uracil by
. gondii tachyzoites in RAW 264 cells pretreated with adsorbed
and unadsorbed lectin-induced rat TIF-containing
supernatant fluids diluted 1:2.
Lectin-induced
TIF-containing supernatant fluid was adsorbed by
CTLL2C1 ?5 cells and assayed for its ability to
inhibit intracellular Toxoplasma replication.
RAW 264 cells were cultured in the appropriate
control (first bar graph), unadsorbed lectininduced TIF (second bar graph) or adsorbed lectin
induced TIF (third bar graph), prior to infection
Bars represent SEM radionucleotide incorporation
for triplicate cultures.
66
Gel-Filtration
Supernatant fluids prepared from lectin-stimulated
rat s plenocy t es were fractionated by gel-filtration using
Sephadex G-100 column chromatography.
IL2 activity,
tested for its ability to induce proliferation of the
IL2-r esponsiv e T-c ell line, CTLL2C17 5, was found to be
present in column fractions 28-29 which eluted from
Sephadex G-100 in the molecular size range of 28,000 to
30,000 daltons.
TIF activity, tested for its ability
to inhibit T oxoplasma replication in tachyzoite-in feeted
RAW 264 cells, was found to be present in column fractions
22-23,
24-25, and 26— 27 which eluted from Sephadex G-100
in the molecular size range of 39,000 to 58,000 daltons
with peak activity at 53,000 daltons (Fig. 10).
Results
from this microassay were expressed as percent inhibition
of
-uracil incorporation by tachyzoite-in f ected RAW
264 cells cultured in column fractions 22-39 versus [^H]uracil incorporation by infected cells cultured in control
supernatant fluid.
In the presence of partially purified TIF (column
fractions 22-23, 24-25, and 26-27) diluted 1:2 in 0.8% HIB,
tachyzoite-infected RAW 264 cells incorporated 3,223 +
379' c pm , 1,860 + 536.cpm and 3,754 + 556 c pm of [
]- ur ac il,
respectively, and appropriately treated control cells
incorporated 7,316 + 841 cpm.
Fraction Number
Fig. 10.
IL2 activity (#--- #) and TIF activity (#-- -#) assayed after Sephadex
G-100 column chromatography of lectin-stimulated rat splenic super­
natant fluid.
IL2 was assayed for its ability to induce proliferation
of CTLL2C1J5 cells.
TIF was assayed for its ability to inhibit the
intracellular replication of _T. gondii in RAW 264 cells.
Tachyzoiteinfected RAW 264 cells pretreated with unfractiona^ed lectin-prepared
supernatant fluid incorporated 3,432 + 67 c pm of [ H ]-uracil (53%
inhibition), whereas infected cells pretreated with the appropriate
control supernatant fluid incorporated 7,316 + 841 c pm.
68
Infected cells cultured in the presence of partially
purified IL2 (column fractions 28-2 9) diluted 1:2 in
0.8% HIB incorporated 6,351 + 577 c pm of [^H]-uracil.
Incorporation of [^H]-uracil by tachyzoite-infected
RAW 264 cells treated with column fractions 30-39 was
similar to that obtained for cells treated with partially
purified IL2 (column fractions 28-29).
The results
obtained from this microassay using fractionated lectinprepared supernatant fluid indicated that partially
purified IL2 was unable to inhibit parasite replication
in RAW 264 cells.
Treatment of Mice with TIF-Containinq Supernatant Fluids
Toxoplasma tachyzoites were demonstrated in each mouse
where infection was diagnosed histologically.
Neither
immune TIF nor lectin-induced rat TIF conferred protection
to mice inoculated with 100 infectious oocysts when com­
pared with control groups (Table 8).
Infected mice
treated with immune TIF-containing supernatant fluid
survived 10.5 (mean) days, whereas infected mice treated
with the appropriate control supernatant fluid survived
10.8 (mean) days.
Groups of infected mice treated with
either lectin-induced TIF or the appropriate control
supernatant fluids, survived 10.2 (mean) days, whereas
the medium control group survived 11.3 (mean) days.
69
Table 8.
Effect of TIE-containing supernatant fluids on
Toxoplasma in fectivity in mice inoculated with
T_. gondii oocysts.
Mean survival
time (days)
Treatment
% Infegted
mice
Immune TIE
10.5
100
Immune TIE control
10.8
, 100
L ectin-in dueed rat TIE
10.2
100
Lectin-induced rat TIE Control
10.2
100
Medium control
11.3
100
a
'
.
Six mice per group were inoculated subcutaneously with
100 infectious oocysts three days after initiation of
treatment.
70
CHAPTER 5
DISCUSSION
Several investigators
(54,97,101)
observed that immune
and nonimmune peritoneal mouse macrophages activated in
vitro with supernatant fluids prepared from antigen- or
Iectin-stimulated lymphocytes display an enhanced capacity
for inhibiting the intracellular replication of Toxoplasma
gondii, compared with the same cells cultured in control
supernatant fluids.
The present study extends these ob­
servations using a homogeneous macrophage-like cell line,
RAW 264, cultured in similar supernatant fluids and in­
fected with
gondii.
Use of this cell line has proven
a helpful tool for studying Toxoplasma infection of macro­
phages and has led to the development of a rapid, repro­
ducible, and accurate microassay, based upon tritiated
uracil incorporation by intracellular organisms,
for
evaluating the capacity of lymphok in e-'acti vat ed phagocytic
cells to inhibit replication of this obligate intracellular
parasite.
In previous studies, it was reported that antigenstimulated lymphocytes from Toxoplasma-immune mice
elaborate a soluble factor, TIE (55), which is capable
of inhibiting the intracellular replication of T_. gon dii
71
within immune and nonimmune peritoneal mouse macrophages
(54,97,101).
This lymphokine, with a reported molecular
weight of 38,000 to. 55,000 daltons (79,103), can also be
generated by stimulating murine lymphocytes with the
T-cell lectin, concanavalin A (Con A) (102).
Supernatant fluids prepared from antigen-stimulated
lymphocytes of Toxoplasma-immuhe animals were shown to
contain a variety' of biologically active substances, other
than T I F , that are also effective in inhibiting %. gondii
replication within host cells (13,104). For example,
Shirahata and Shimizu (104) reported that in response to
antigenic stimulation, spleen cells from Toxoplasmainfected mice elaborate a soluble factor which inhibits
vesicular stomatitis virus (USV) and Mengo virus infection
in L cell cultures.
This factor, which was found to be
immune interferon, was also shown to inhibit the growth of
Toxoplasma within nonphagocytic mouse embryonic fibro­
blasts and was reported to have a molecular weight of
approximately 46,000 daltons (104).
In addition, the
studies by Chinchilla and Frenkel (13) demonstrated that
in vitro reactivation of lymphocytes from Toxoplasma-immune
hamsters with Toxoplasma lysate antigen results in the pro­
duction of a soluble factor which, like TIF, is capable of
inhibiting the intracellular replication of T_. gondii
within immune and nonimmune peritoneal hamster macro­
phages.
However, based upon its low molecular weight of
72
4,000 to 5,000 daltbns, and its ability to inhibit
Toxoplasma replication in hamster kidney cell and fibro­
blast cultures (13), this soluble lymphocyte product is
called Toxoplasma-immune mediator and is thus distinguished
from T I F .
In the present study, another T-cell derived product,
known as interleukin
2 (IL2 ) (1,35,56), was also specu­
lated to be present in supernatant fluids prepared from
antigen- and lectin-stimulated spleen cells, since it was
previously shown that the production of IL2 occurs under
conditions almost identical to those required for the
production of TIF (5,35,56,75).
This investigation was
carried out in an attempt to determine whether IL2 was
present in TIF-containing supernatant fluids and, if
detected, whether it had an effect on _T. gondii multipli­
cation within nonimmune peritoneal mouse macrophages and/or
RAW 264 cells.
The data presented in this report indicate that IL2 .
was present in TIF-containing supernatant fluids prepared
from Toxoplasma-imm.u ne mouse splenocytes stimulated with
Toxoplasma lysate antigen and from nonimmune rat spleno­
cytes stimulated with the T-cell lectin, Con A.
When
supernatant fluids were examined for the presence of
IL2, a considerable amount of IL2 activity was detected,
as assessed by its ability to induce proliferation of the
IL2-responsive T-cell line , CTLL2CI?5 (Table 4).
73
Conversely, adsorption of supernatant fluids with CTLL2C1/5
cells removed all detectable levels of IL2 activity
(Tables
6,7) .
Since these observations demonstrated that IL2 was
elaborated from antigen- and lectin-stimulated spleen
cells, supernatant fluids were assayed for their ability
to inhibit intracellular Toxoplasma replication within
nonimmune peritoneal mouse macrophages and RAW 264 cells.
When ant!toxoplasma activity was assessed by direct micro­
scopic observation of Giemsa-stained preparations (Tables
2,3) and/or tritiated uracil incorporation into nucleic
acids of intracellular parasites (Figs. 1,3,4,5),
it was
observed that both antigen- and lectin-stimulated splenic
supernatant fluids significantly inhibited Toxoplasma
replication within these cells.
These findings were con­
sistent with the report by several investigators (97,101,
102) that non immune peritoneal mouse macrophages exposed
to supernatant fluids prepared from antigen- or lectinstimulated splenocytes display an enhanced capacity to.
inhibit Toxoplasma replication.
However, although
Toxoplasma inhibition was noted in both nonimmune perito­
neal mouse macrophages and RAW 264 cells, the results from
these experiments did not demonstrate that IL2 was re­
sponsible for. the inhibition observed, since TIF was also
present in antigen- and lectin-prepared supernatant fluids.
74
Experiments were subsequently performed to determine
whether removal of IL2 activity from antigen- and lectinprepared supernatant fluids by CTLL2C175 cells had an
effect on these supernatant fluids to inhibit intracellular
I ox oplasma replication within RAW 264 cells.
The results
from these experiments showed that although tachyzoiteinfected cells exposed to adsorbed supernatant fluids
inhibited parasite replication, the degree of inhibition
was not as great as that observed in infected cells exposed
to nona dsorbed supernatant fluids (Figs. 8,9).
It appeared
that some of the TIF activity present in antigen- and
lectin-prepared supernatant fluids was also adsorbed by
CTLL2C175 cells.
However, since adsorption of supernatant
fluids with C T L L 2C1 75 cells removed all detectable levels
of IL2 activity and only minimal amounts of TIF activity,
these experiments were considered in conclusive.
To determine whether IL2 alone was capable of
inhibiting Toxoplasma replication, additional experiments
were performed using TIF-free supernatant fluid prepared
from Con A-stimulated FS6-14.13 cells, an IL2-producing
T-c el I hybridoma.
When RAW 264 cells were cultured in.
FS6-deriv ed supernatant fluid and infected with
gondii,
a marked proliferation of organisms was noted (Fig. 7)
compared with the same cells cultured in supernatant fluids
prepared from antigen- and lectin-stimulated spleen cells
75
(Figs. 3,4,5).
These results strongly suggested that
TIF and IL2 were two distinct and unique molecules.
Since the use of supernatant fluid prepared from ■
Con A-stimulated FS6-14.13 cells may have represented an
artificial means of examining the effect of IL2 on tachyzoite-infected cells, supernatant fluid prepared from
Iect in-stimulated rat splenocytes was fractionated by
gel-filtration using Sephadex G-100 column chromatography
and then assayed for IL2 and TIF activity.
When fractions
were assayed for their ability to inhibit Toxoplasma repli­
cation in RAW 264 cells,
it was noted that the fraction
which had a MW corresponding to that of murine IL2 induced
proliferation of the IL2-responsive T-c ell line, CTLL2C175,
but had no effect on _T. gondii replication (Fig. 10).
These findings were consistent with the results obtained
from the previous experiment using FS6-derived supernatant
fluid in which no inhibition of Tv gondii replication was .
observed, indicating further that TIF and IL2 were two
separate molecules.
Although IL2 was unable to directly inhibit Tv qondii
replication in parasitized cells, the elaboration of both
TIF and IL2 from specifically and nonspecifically stimu­
lated T lymphocytes may be advantageous to the host
following Toxoplasma infection.
For example, while TIF
is effective in inhibiting Toxoplasma replication in host
cells (54,97,101),
IL2 functions to provide the stimulus
76
necessary for proliferation of helper and cytotoxic
I lymphocytes (71,107).
The T-c ell proliferative response
is comprised of a complex series of interactions between
at least three cell types: macrophages, IL2-produc er cells,
and IL2-responsive cells (105).
The signals required for
the generation of T-c ell proliferation and subsequent
maturation to an effector cell function involve lectin/
antigen cell membrane binding, release and response to
ILl, a monokine, and release and response to IL 2, a
lymphokine (57,71,105).
Since IL2 ultimately mediates
the proliferative response and is the end product of these
reactions, it is the regulatory factor that finally
determines the magnitude of T-c ell clonal expansion.
Antigen-specific helper and cytotoxic T-c elI clones,
which are regulated by IL 2 (71,107), may play a role in
limiting _T. gondii infection.
Since helper T-c el Is are
required for the production of antibody'to T- depen dent
antigens (64), it seems reasonable to speculate that the
interaction of these cells with specifically-sensitized
B-c ells results in the production o f.T_. qondii-specific
antibody.
Although the presence of antibody, is not
sufficient for protection against %. gondii infection,
as shown by the ability of the parasite to persist in the
tissues of the infected host in the presence of very high
antibody titers (27), it nevertheless may aid to limit
further parasite replication within host cells.
Recent
77
work showed that the ability of macrophages to kill
infective tachyzoites is greatly increased if the parasites
are first exposed to antibody and complement (5,54).
Thus,
opsonization of Toxoplasma with specific antibody and
subsequent digestion by phagocytic cells, may represent
one mechanism by which
parasite number is reduced in the
infected host.
It is well established that cytotoxic T lymphocytes
directly participate in the cell-mediated immune response
by killing target cells which express specific surface
antigens (7,35).
However, the interaction between these
cells is restricted to the host's major histocompatibility
complex (MHC); effective killing by cytotoxic T-c ells is
dependent upon the presence of MHC-directed antigens on
the surface of the target cell (117).
Since MHC- directed
antigens are probably not on the surface of, Toxoplasma
tachyzoites (11), cytotoxic T-cells are probably not
involved in direct killing of the organisms.
Cytotoxic
T-cells may indirectly limit parasite infection by
interacting with Tv gondii-directed antigenic
determinants
which may.be present on the surface of the infected host. .
cell.
Previous studies demonstrated that IL2 also functions
to induce the proliferative response of natural killer
78
(NK) cells (16,17), a subpopulation of lymphoid cells that
play an important role in immunosurveillance by mediating
natural resistance against tumors and certain viral and
microbial diseases (43).
Ortaldo and Herberman (83)
recently reported that NK cells can recognize several
widely distributed antigenic specificities, and that such
recognition is clonally 'distributed.
Furthermore, these
investigators suggested that recognition by NK cells does
not appear to require expression of products of the MHC
on target cells.
Although the mechanisms of killing by
NK cells are unclear, it was suggested that binding to
target cells via T-cell-like receptors is first required,
followed by the lytic event (39,109).
Several investigators (42,43,90) suggested that NK
cells are involved in resistance against some types of
microbial infections and tumors.
For example, Ruebush and
Burgess (90) found a correlation between increased levels
of NK activity and resistance of mice to the protozoan,
Babesia microti.
Similarly, Hauser et a l . (42) reported
that acute infection of mice with _T. gondii results in
enhanced NK cytotoxicity ijn vitro for YAC-I tumor target
cells.
These investigators also found that augmentation
of NK activity is macrophage-dependent since depletion
of macrophages by in_ vivo administration of silica com­
pletely abrogates Tv g on dii-in due ed NK cytotoxicity.
Other investigators demonstrated that NK cells will
79
proliferate in response to IL2 (16,17), a lymphokine whose
production is also macrophage-dependent (105).
Thus, it
appears that the elaboration of IL2 by specifically
stimulated amplifier T-c.ells is responsible for the
augmented NK activity observed in _T. qondi i infected mice.
In addition,
this IL2-induced augmentation of NK activity
might explain the reported resistance of Toxoplasma
infected mice to various infectious agents (34,91) and
tumors (44,45,46).
Macrophages are the body's chief defense against
foreign particulate matter and chronic infection (68).
Results of previous studies demonstrated that macrophages
respond to certain infections with an adaptive increase
in defensive capacities (60,110).
Enhancement of macro­
phage function during infection has an immunologic basis
(61) and often involves lymphocytes and their products
(i.e., lymphokine)
(62).
Activated macrophages obtained
from animals during a certain stage of infection were
reported to become larger, spread out more readily on
glass, contain more mitochondria and lysosomes, and display
a greater phagocytic ability than macrophages obtained
from normal animals
(63).
Of greater importance is the
fact that such activated cells have an enhanced bac­
tericidal capacity which is effective even against
organisms antigenically unrelated to those infecting the
host (61).
80
A number of investigators (54,97,101,102) successfully
activated macrophages of laboratory animals and humans to
inhibit or kill T_. gondii by exposing the macrophages in
vitro to supernatant fluids prepared from antigen- or
lectin-stimulated I lymphocytes.
Furthermore,
iin vitro
reactivation of immune lymphocytes with specific antigen
was reported to result in the production of a soluble
mediator which is effective in conferring protection to
animals challenged _in vivo with Besnoitia J ellisoni, a
protozoan related to _T. gondii (13).
According to the report by Chinchilla and Frenkel
(13), a.single intraperitoneal injection of 2.0 ml of
supernatant fluid prepared from antigen-stimulated
lymphocytes of Besnoitia-immune hamsters significantly
delays the death of nonimmune hamster's challenged sub­
cutaneously with 10 to 100 infective Bv j ellisoni tachyz'oites when compared with serum from normal animals.
These investigators also found that the soluble factor
present in active supernatant fluid, which they call
Besnoitia-immune mediator, affords hamsters better pro­
tection against infection than high-titer Besnoitia
antiserum.
They suggested that repeated injections of
mediator might increase its effectiveness _in vivo which
indicated that further experimentation was required.
In the present study, TIF-containing supernatant
fluids were also speculated to afford mice protection
81
against T_. gondii infection since Besnoitiarimmune mediator
was reported to be effective against EL j ellisoni infection
However, although mice were treated daily, for greater
than two weeks, with intraperitoneal injections of 1.0
ml of supernatant fluids prepared from antigen- and lectinstimulated splenocytes, no degree of protection against
X* gondii infection was observed when compared with mice
treated with control supernatant fluids (Table 8).
This
lack of protection may have reflected the volume of super­
natant fluid used for injection, since Chinchilla and
Frenkel (13) found that a 2.0 ml injection of active super­
natant fluid is effective in conferring immunity to
B esnoitia infection.
It may be possible that antigen-
and lectin-stimulated splenocytes of Toxoplasma-immune
and nonimmu ne animals, respectively, elaborated smaller
quantities of lymphokine compared with antigen-stimulated
lymphocytes of Besnoitia-immune animals, thereby de­
creasing the concentration of mediator present in the
supernatant fluid.
are indicated.
Additional experiments in this regard
The development of an assay to quanti­
tatively determine the level of TIF activity present in '
supernatant fluid seems appropriate to determine whether
supernatant fluids containing concentrated levels of TIF
are capable of conferring immunity to Tv gondii infection.
The mechanism by which phagocytic cells inhibit
intracellular Tv qondii replication is not clearly under­
stood.
Jones and coworkers
(55) reported that Toxoplasma
inhibition in macrophages is associated with increased
levels of cAMP and decreased levels of cGMP.. They found
that cells from immune animals treated with or without
the addition of lymphokine show a marked increase in
cAMP and a decrease in cGMP.
Nonimmune macrophages
stimulated with an inflammatory agent, such as heart
infusion broth, and then cultured with lymphokine. were
reported to show a similar and significant change in
nucleotide levels., whereas macrophages treated with heart
infusion broth alone or lymphokine show no such increase.
These investigators concluded from their studies that
those conditions with significant changes in cyclic
nucleotide levels are those which also are associated
with inhibition of Toxoplasma multiplication.
Oxidative metabolites were also suggested to play
a significant role in the antimicrobial activity displayed
by macrophages (75).
al.
Murray and Cohn (73) and Murray et
(74) reported that Tv gondii is susceptible to oxygen
intermediates generated by the partial reduction of
molecular oxygen.
Results of their experiments demon­
strated that although the parasites are resistant to super
oxide anion (Og) and hydrogen peroxide (HgOg), they are
83
readily killed by hydroxyl radical (OH •) and singlet oxygen
(1O 2) as a result of the 0~ - H^O^ interaction (73).
Several investigators (73,74,75) reported that
phagocytic cells display varying capacities to generate
oxygen intermediates, as judged by extracellular release
of H 2O 2-
Macrophages from nonimmune mice were found to
release small amounts of HgO2 and allow unrestricted
multiplication of intracellular T_. gondi i .
Macrophages
from chronically infected, Toxoplasma-immune mice were
reported to release four times more H 2O2 and display
significant toxoplasmastatic activity.
When macrophages
from immune-boosted mice were examined for their ability
to generate oxygen intermediates, it was shown that these
cells release twenty-five times more H 2O2 than nonimmune
cells and rapidly kill most of the parasites.
Thus, only
activated macrophages have the capacity to generate potent
oxygen intermediates which serve as important effector
molecules in both the destruction and growth inhibition
of %. gon dii .Murray and Cohn (75) examined the antitoxoplasma
activity and oxidative capacity of peritoneal mouse macro­
phages stimulated jm vivo by inflammatory and immunologic
agents and iji vitro by lymphokines.
These investigators
reported that normal macrophages and those elicited by
in vivo administration of inflammatory agents,. such as
84
thioglycollate and heart in fusion broth, readily, support
intracellular
gon dii replication.
Normal macrophages
were also reported to release four to twenty times less
^2*^2 than cells activated i^n vivo by systemic infection
with Bacille Calmette-Guirin or X* Qondii (75).
When
normal, macrophages and those elicited by inflammatory
agents are activated in. vitro by exposure to antigen- or
lectin-prepared lymphokines,
and
release are
stimulated, but antitoxoplasma activity is not observed
in these cells.
In contrast, nonimmune macrophages
cocultivated with lymphokine plus heart infusion broth
display significant toxoplasmastatic activity.
Murray
and Cohn (75) concluded from their studies that increased
oxidative metabolism is a consistent marker of macrophage
activation and that oxygen intermediates play an important
role in the resistance of activated macrophages to
infection with X* Qon dii.
Since the results of the
present experiment demonstrated that RAW 264 cells co­
cultivated with heart infusion broth plus supernatant
fluid prepared from antigen- or lectin-stimulated spleen
cells inhibited intracellular X* Qondii replication, it
would be of interest to study the effect of oxygen
intermediates on the fate of this pathogen within RAW 264
cells.
Destruction or growth inhibition of X* gondii by
phagocytic cells was suggested to depend upon the
85
interaction of several variables.
Recent studies showed
that these variables include the
I) effective triggering
of the macrophage respiratory burst (116); 2) magnitude
of the oxidative burst (74); 3) generation of toxic oxygen
intermediates beyond the production of
and
(74,75);
and 4) activity of competitive intracellular mechanisms
that scavenge 0~ and/or H 2O2 (76).
Murray and coworkers.
(76) suggested that the latter inhibitory systems might
result in diminished generation of products of O2 - H2O2
interactions, such as OH • (33,89), a potent oxidant that
is both bactericidal and toxoplasmacida I (51,73,74).
Normal peritoneal mouse macrophages and those elicited
by inflammatory agents were demonstrated to display little
oxidative activity
(74,75).
In fact, these cells were
reported to allow unrestricted multiplication of intra­
cellular
X- gondii (74,75).
The ability of this obligate
intracellular parasite to parasitize these cells was sug­
gested to result from ineffective triggering of the
oxidative respiratory burst of nonactivated cells (75,116)
I
and resistance of the parasite itself to the toxicity of
either 0” or H2O 2 (73).
Furthermore, the presence of
endogenous 0~ and H2O 2 scavenging enzymes, such as super­
oxide dismutase (SQD) , glutathione peroxidase (GPO), and
catalase, which were reported to function within X- gondii
as antioxidant protective mechanisms, may reduce the
effectiveness of phagocyte antimicrobial activity (76).
86
More recently, Murray
(77,78) reported that nonimmune
peritoneal mouse macrophages and lymphokine-treated J774G8
cells, a homogeneous macrophage-like cell line, display
enhanced generation of HgOg anc* inhibit replication of
L eishmania donovani and ,Leishma nia tropica, the etiologic
agents of vise era I and cutaneous leishmaniasis,
tively .
respec­
Ingestion of parasites readily triggers the
oxidative burst of infected cells, and results in the
generation of HgOgi a toxic oxygen intermediate to which
Leishmania was found to be highly susceptible.
In contrast,
unstimulated 077408 cells which are also infected with
these parasites,
fail to generate an oxidative burst and
readily support their intracellular replication.
It would
be of interest to investigate the events occurring after
infection of unstimulated RAW 264 cells with L eishmania
to determine whether these phagocytic cells possess
oxidative capacities similar to those observed in nonimmune
peritoneal mouse macrophages,
Interactions between intracellular %. gondii and
host cells are complex.
The parasites induce marked
changes in the function of.infected macrophages and some­
how alter the immunologic response of the host.
Opson­
ization of Toxoplasma with specific antibody elicited by
the infected host leads to enhanced microbial killing
and subsequent digestion by phagocytic cells.
Also,
87
soluble lymphocyte products appear' to interact with
macrophages by creating an environment which kills or.
inhibits replication of this obligate intracellular
parasite.
Although the killing or, growth inhibiting
mechanisms remain unknown, experimental results from the
present study indicate that TIF plays an important role
in limiting T_* gondii infection.
The RAW 264 microassay
has proven to be a useful method to study the effect of
this soluble lymphocyte product on Toxoplasma replication
within parasitized cells.
88
CHAPTER 6
SUMMARY
A rapid, reproducible, and accurate microassay using
a homogeneous macrophage-like cell line, RAW 264, was
developed to study the effects of soluble lymphocyte
products on Toxoplasma gondii replication within para­
sitized cells.
RAW 264 cells were cultured for 19.5 h in
supernatant fluids prepared from specifically and non specifically stimulated T lymphocytes and infected with
virulent RH strain Toxoplasma tachyzoites for 30 to 90 min
Sixteen to 20 h after the addition of culture medium,
antitoxoplasma activity was assessed ,either by direct
microscopic observation of Giemsa-stained preparations or
by tritiated uracil incorporation into nucleic acids of
intracellular parasites.
Supernatant fluids prepared from antigen- and lectinstimulated murine spleen cells effectively inhibited intra
cellular j[. gondii replication within RAW 264 cells and
non immune peritoneal mouse macrophages.
However, mice
repeatedly injected with the same supernatant fluids were
afforded no protection against Toxoplasma infection when
challenged in_ vivo with infectious oocysts.
89
The ability of antigen- and lectin-prepared super­
natant fluids to inhibit T_. gondii replication within,
tachyzoite-infected cells and to induce proliferation of
the interleukin 2 (IL2)-responsive T-cell line, CTLL2C175, .
indicated that both Toxoplasma inhibitory factor (TIF) and
IL2 were present in the same unfractionated supernatant
fluids.
To determine whether IL 2 alone was also capable
of inhibiting Toxoplasma replication within parasitized
cells, a series of experiments were performed.
Results
demonstrated that adsorption of antigen- and lectin-prepared
supernatant fluids with CTLL2C175 cells removed all de­
tectable levels of IL2, while having little effect on TIF.
Elaboration of IL2 from concanavalin A (Con A)-stimulated
FS6-14.13 cells induced proliferation of CTLL2C175 cells,
but failed to inhibit Ti. gondii replication within tachy­
zoite-infected cells.
Fractionation of lectin-prepared
supernatant fluids by gel-filtration using Sephadex
G-100 column chromatography showed that TlF and IL2
eluted in two separate peaks of activity.
TIF activity,
as assessed by its ability to inhibit Toxoplasma replica­
tion within RAW 264 cells,
eluted from Sephadex G-100 in
a broad peak in the region of 39,000 to 38,000 daltons.
IL 2 activity, as assessed by its ability to induce pro­
liferation of CTLL2C175 cells,
eluted in the molecular
size range of 28,000 to 30,000 daltons.
Thus, although
',I
90
both TIF and IL 2 were found to be present in the same
antigen- and lectin-stimulated splenic supernatant fluids,
experimental results suggest that these soluble lymphocyte
products are two separate molecules.
91
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