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i
rHESlg ‘ LXEIARY

 

This is to certify that the

dissertation entitled

Mechanisms of Alteration of Cellular Immune

Responsiveness during Experimental Trxpanosoma

 

cruzi Infection.

presented by

James Ray Maleckar

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in _Mj_cmbj_n_]_o_gy and
Public Health

yumuh/ jc’zJ

Major pro ssor

Date May 20, 1983

MSU is an Affirmative Action/Equal Opportunity Institution

042771

 

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RETURNING MATERIALS:
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.5 1‘
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’ “1' 1.". '
‘ - l

 

 

MECHANISMS OF ALTERATION OF CELLULAR IMMUNE RESPONSIVENESS
DURING EXPERIMENTAL Trypanosoma cruzi INFECTION

 

By

James Ray Maleckar

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1983

IVs-3733

ABSTRACT

MECHANISMS OF ALTERATION OF CELLULAR IMMUNE RESPONSIVENESS DURING
EXPERIMENTAL TRYPANOSOMA CRUZI INFECTION.

 

BY
JAMES RAY MALECKAR

Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma

 

5:351, usually presents an acute and a chronic phase. Reports of normal
immune responses have often conflicted with other studies which have described
an immunologically deficient state in Chagas' disease. A survey of the
literature on this subject has suggested that immunosuppression is limited to
the acute phase. The present work delineates the kinetics of the suppression
by examining the host's ability to produce a delayed-type hypersensitivity
reaction both 13 vitrg and ifl.!i££9.t° I, 95351 antigens in the

mouse model system of Chagas' disease. The measured parameters, footpad
swelling and inhibition of macrophage migration, did not differ significantly
from those of uninfected mice during the initial 40 days postinfection.
Removal of Lyt 2.1-bearing cells from an acute mouse spleen cell suspension
did not restore responsiveness to that population. Furthermore, addition of
cells from acutely infected mice to chronic mouse spleen cells did not affect
the ability of the latter to respond to stimulation with the trypanosomal
antigen. Thus, this work indicated that suppressor T cells were not involved
in production of immunosuppression. The ability of the parasite to modulate

mitogen-induced lymphoproliferation was next examined. The responses of

normal mouse spleen cells to either concanavalin A (Con A) or a bacterial
lip0polysaccharide (LPS) were reduced in the presence of sufficient numbers of
either culture (epimastigote) or virulent bloodstream (trypomastigote) forms
of I, 91351. This suppressive action was found to be dependent on the
parasite dose and not to be due merely to removal of the mitogen by absorption
onto the parasite surface. Non-living preparations of T. cruzi were shown

to be suppressive, indicating that parasite viability was not a requirement
for production of suppression and that the phenomenon was not due to
competitive consumption of essential nutrients by the parasites. Suppressed
lymphoproliferation was observed when only the non-adherent spleen cells from
uninfected mice were incubated with the trypanosomes. Treatment of adherent
spleen cells with the parasite had no significant effect on the ability of
these cells to support lymphocyte responses to mitogens. These results
demonstrate that immunosuppression in an infected mammalian host is
restricted to the acute phase of Chagas' disease and suggest that the parasite
itself contributes to this effect by modulating lymphocyte function by an as

yet undefined mechanism.

ACKNOWLEDGEMENTS

I would like to express my deepest gratification to Dr. Kierszenbaum
whose wisdom and patience has played a major part both in my development
as a scientist and as a person. Although the path I followed was not
always the straightest or the quickest his enthusiasm and input never
let me lose sight of my goal.

I would like to express my thanks to my wife, Veronica. Even though
I received the degree, I feel that she earned it as well as I did. Her
support, patience, and understanding as well as her humor and willing-

ness has made the journey much easier to travel.

11'

TABLE OF CONTENTS

Page

List of Figures ................................................. v
List of Tables ..... ............................................ vii
Abbreviations .. ................................................ viii
Introduction .... ............................................... 1
Literature Review .............................................. 4
Natural Immunity ........................................... 4
Humoral Inmunity .......................... 6
Non-Specific Antibody Responses ........................... 6
Specific Antibody Responses ............................... 6

Cell Mediated Immunity (CMI) ................................ 7
Protective Effects of CMI....... ........................... 7
Delayed Type Hypersensitivity (DTH) ............... . ....... 8
Cytotoxicity Mechanisms ............ ....................... 8
Destruction of T. cruzi by MacrOphages .................... 9
Suppression of CMI .......................................... 10
Altered Responsiveness to Mitogenic Stimulation ........... 10
Depressed Drug-Induced Contact Sensitivity ............... . 11
Mechanisms of Inmunosuppression ............................. 11
Suppressor T Lymphocytes ... ............................... 11
Adherent Suppressor Cells ................................. 11
Parasite-Induced Suppression .............................. 12

List of References .......................................... 13

iii

Page

CHAPTER I. VARIATIONS IN CELL-MEDIATED IMMUNITY T0 TRYPANOSOMA
CRUZI DURING EXPERIMENTAL CHAGAS' DISEASE

 

Abstract ................................................... 21
Introduction ............................................... 23
Materials and Methods ...................................... 24
Results .................................................... 28
Discussion ................................................. 39
References .............. . ................ ...... ............ 43

CHAPTER II. SUPPRESSION 0F MOUSE LYMPHOCYTE RESPONSES TO MITOGENS
IN VITRO BY TRYPANOSOMA CRUZI

 

Abstract ................................................... 46
Introduction ............................................... 47
Materials and Methods ...................................... 48
Results . ................................................... 51
Discussion ................................................. 63
References ................................................. 66

CHAPTER III. INHIBITION OF MITOGEN INDUCED PROLIFERATION OF MOUSE
T AND B LYMPHOCYTES BY BLOODSTREAM FORMS OF TRYPANOSOMA

 

ggygl
Abstract ............................................ . ...... 69
Introduction ............................................... 70
Materials and Methods ...................................... 71
Results .................................................... 75
Discussion ................................................. 87
References . ................................................ 90
SUMMARY AND CONCLUSIONS ........................................ 92
APPENDIX ....................................................... 95

iv

Figure

LIST OF FIGURES

Chapter I

Course of T. cruzi infection in CBA/J
mice ........................ ........................

Delayed-type hypersensitivity reaction elicited
by STA during the course of experimental T.
cruzi infection........................... ...........

Variations in the % MI during the course of
experimental I. cruzi infection............ ..... .....

Effects of mixing chronically infected mouse
cells with cells from either normal or acutely
infected mice on the 1 MI............................

Chapter II

Effect of addition 0f.I- cruzi epimastigotes
to spleen cell cultures on their ability to
respond to conAand LPSOOOOOOOOOOOOOOOOOOOOOOOOOO...

Suppressive effect of T, cruzi epimastigotes
at varying concentrations of Con A............. ......

Suppressive effect of T. cruzi epimastigotes
at varying concentrations of LPS............... ......

Kinetics of suppression of Con A- and LPS-induced
responses by I. cruzi epimastigotes..................

Chapter III

Dose dependence of trypomastigote-induced
inhibition of mouse lymphocyte responses to
C0” A0... OOOOOOOOOOOOOOO O 000000000000 0.... ..........

Dose dependence of trypomastigote-induced
inhibition of mouse lymphocyte responses to

LPS ....................................... .........

.1. cruzi induced inhibition of mouse
Lymphocyte responses to Con A over a full
dose range ......OOOOOOOOOOOOO ....... O 0000000000 O...

 

29

31

34

36

53

54

56

60

76

77

79

vi

Figure Page
4 T. cruzi-induced inhibition of mouse

 

Tymphocyte responses to LPS over an extended
dose range.‘......OOOOOOOOOOI.........OOOOCOCOOOOOOOO 80

5 Kinetics of I. cruzi-induced inhibition of
mouse lymphocyte responses to Con A and LPS ...... ... 86

Table

LIST OF TABLES

Page
Chapter I
Effect of removal of Lyt 2.1-positive
lymphocytes on the responsiveness of
acutely and chronically infected mouse
cells in the MIF test............... ........ ......... 38

Chapter II

Mitogenic capacity of Con A and LPS before
and after absorption with epimastigotes.............. 58

Suppressive effects of preparations of
nonliving T. cruzi epimastigotes (FTE) ....... ........ 62

Chapter III
Mitogenic capacity of solutions of Con A
before and after absorption with trypomastigote
forms 0f 1. CrUZiOO....0I...O....-.....IOOOOOOOOIOOOO 82

Inhibition of Con A- and LPS-induced mouse
lymphocyte responses by STC.......................... 83

Appendix
The effect of addition of varying numbers of
treated or untreated adherent cells on the non-

adherent cell response to Con A and LPS in the
presence or absence of I. cruzi....................... 96

vii

ADCC
CMI
Con A
cpm
CSA
DTH
FTE
ip
LPS
MEM
MEMS

MIF
NK
PBS
PHA
p.i.
STA
STC

.1. cruzi

ABBREVIATIONS

Antibody dependent cell-mediated cytotoxicity
Cell mediated immunity

Concanavalin A

Counts per minute

Crude sonicated antigen (Kuhn, 1973)

Delayed type hypersensitivity

Freeze-thawed epimastigote antigen
intraperitoneal

Bacterial lipopolysaccharide

Minimal essential medium

Minimal essential medium supplemented with 10%
fetal calf serum

Macrophage inhibition factor

Natural killer cells

Phosphate buffered saline

Phytohemagglutinin

Post infection

Soluble trypanosome antigen (epimastigote origin)
Sonicated I, 23251 preparation (trypomastigote
origin)

Trypanosoma cruzi

 

viii

INTRODUCTION

Trypanosoma cruzi, a hemoflagellate protozoan first described by

 

Carlos Chagas in l909 (1,2), is the causative agent of American
trypanosomiasis or Chagas' disease. It is primarily found in rural and
underdeveloped areas and human cases have been reported from Argentina to
the southern United States (3). Currently at risk are approximately 50
million people whereas it has been estimated that over 7 million people
have previously contracted the disease (4). Cardiopathology (mainly
chronic myocarditis), megacolon, and megaeSOphagus are not uncommon
manifestations of the chronic phase of Chagas' disease (5, 6). In endemic
regions of Chagas' disease, chronic myocarditis is the leading cause of
heart failure and sudden death (7). Therefore, it is not surprising that
Chagas' disease represents a major health problem in vast areas of the
American continent.

.1..ggggi is transmitted to man and other mammals by several
species of blood-sucking reduviid bugs. In the invertebrate host, the
parasite multiplies in the epimastigote form while migrating down the gut.
In the hind gut, epimastigotes transform into the infective metacyclic
trypomastigote stage and are passed out with the feces. Man acquires the
parasite by rubbing contaminated feces into broken or intact skin, mucosa,
or the lesion left by the bite of the reduviid bug. Organisms invade
local tissue cells and multiply intracellularly as amastigotes. This form
then transforms into the trypomastigote stage just prior to the rupture
of the cell. Following lysis, trypomastigotes are transported via the
bloodstream, body fluids and lymphatics thus reaching other types of

tissues and cells. The cycle is completed when trypomastigotes in the

l

blood are ingested by the vector.

Chagas' disease often starts with an acute, subacute or subclinical
acute phase in which the parasites may or may not be readily detectable
in the circulation. Any of these forms of the disease is followed by
the chronic phase in which the parasites are frequently not detected by
routine screening methods. The immunology of Chagas' disease has been
the subject of several reviews in recent years (8-11). Whereas a more
detailed discussion of past and current progress in this area will be
presented in the Literature Review, below is a brief outline of the
approaches made in the present research program.

In the host, I._grggi is both exposed to and sequestered from
the effector mechanisms of the immune system. Resulting from these
conditions is a delicate balance between parasite destruction and
survival. During the acute phase of the disease relatively low levels
of serum antibodies occur which are specific for I. Egggi that may
play an important role in controlling the course of the infection.
Concomitantly, the humoral response to other antigens and the cellular
immunity are severely suppressed. Responses to both 1. grggi and
unrelated antigens return to levels comparable with those of uninfected
individuals during the chronic phase of the disease. Our understanding
of the mechanisms involved in host resistance to the parasite,
production of immunosupression and control of the infection is far from
being complete.

At the time that this research program was undertaken there was
confusion in the literature as to the meaning of impaired immunological

alterations occurring during experimental Chagas' disease in the light

of other reports describing normal responsiveness. Careful screening of
the apparently conflicting evidence revealed that suppressed responses
had been documented in experimental models of the acute phase of the
infection whereas normal responses were obtained with chronic chagasic
patients. The possibility that suppression and immune responsiveness
could represent two distinct phases of the disease was construed as the
first working hypothesis to be explored in this work. Results of a
study in which delayed-type hypersensitivity to a crude trypanosomal
antigen was monitored both ig_vivg and jg vitrg_during the

entire course of an experimental I, grggi infection will be

presented in Chapter I (12).

The subject of induction of immunosuppression was addressed in the
second phase of this research. The possible role ofII.Ig§ggi in
modulation and/or impairment of immune responses was examined by using a
protocol in which either the parasite or its products altered
mitogen-induced lymphocyte proliferation. The results of these studies
are presented in Chapters II (13) and III (14).

A final but important consideration in this program was to obtain
information about the cell type(s) that, influenced by the parasite,
contribute to the establishment of the suppressed state. Incubation of
the parasite with the main cell types involve in the mounting of a
lymphoproliferative response and the effect of such treatment on the
response itself was the selected approach. The results of these efforts
will be presented in Chapter IV.

Closing this Thesis will be a Surrmary highlighting the conclusions

derived from this work and their significance.

LITERATURE REVIEW

The complexity of the disease caused by Trypanosoma cruzi,

 

Chagas' disease, has been recognized since Carlos Chagas first reported
its existence as a clinical entity related to an etiological agent (1).
In examining various tissues of chagasic patients, Vianna (16) reported
intense inflammation, including lesions in the heart, in infected
individuals whether or not the parasite was present at the site. Many
cell types were found to be vulnerable to invasion by the parasite,
muscle, reticuloendothelial tissues, and glial cells being the primary
target cells (16-19). Extensive information about parasite localization
in the infected host contrasts with our limited understanding of the
mechanisms of defense against infection.

Natural Immunity

 

I, £2351 has no known mammalian host specificity. It is capable
of parasitizing a wide range of wild and domestic animals but amphibians
and birds are naturally resistant [reviewed by Brener (20)]. Kierszenbaum
_gt.gl. (21,22) demonstrated that avian resistance was antibody
independent but complement dependent. Thus, blood forms of the parasite
were readily detectable in the circulation of chickens previously depleted
of complement by intravenous injections of cobra venom factor 24 hr after
intravenous injection whereas in normal animals the flagellates could not
be detected within a few minutes after inoculation. Lysis of the
trypanosomes by avian serum in vitro required the presence of magnesium

ions but not calcium ions, indicating that the alternative

pathway of complement activation was involved. Similar results were
obtained when trypomastigotes grown in irradiated mice or in tissue
culture were used, clearly indicating that antibodies were not required
(22). Whereas, trypomastigotes are not lysed by normal mammalian sera,
epimastigotes are sensitive to these sera (23,24).

Many non-specific factors affect resistance and susceptibility to
.1..grgzi. Among these are age, sex, and nutritional aspects of the
host along with environmental conditions, such as temperature (reviewed
in 6). Resistance also varies substantially between species of animals
as well as within a given species. Studies performed in inbred strains
of mice have demonstrated variations in the degree of susceptibility to
“I. grgzi infection, ranging from the very susceptible to the highly
resistant (25). Trischmann and Bloom (26) have suggested that host
resistance in mice may be governed by multiple genetic factors not
including those coded for at the major histocompatibility complex (H-2).

Recent evidence has indicated the presence of natural killer (NK)
cells capable of lysing trypomastigotes and unrelated tumor cell lines
in uninfected as well as I} grgzjfinfected mice (27,28). This lytic
activity was demonstrated within 48 hr of infection. Both spleen and
peritoneal exudate cells exhibited antifil..g£gzi lytic activity
which was increased after I, grgzi infection or sensitization with
polyinosinic-cytodylic acid and decreased by treatment with anti-NK 1.2
antiserum plus complement. However, the significance of this activity to

host resistance is not known.

Humoral Immunity

 

Non-Specific Antibody Responses. The acute phase of experimental
Chagas' disease is accompanied by an inability to produce antibodies
against non-related antigens ig_vitrg. Clinton gt El; (29) did
not observe normal production of antibody-forming cells (plaque forming
cells, PFC) following immunization with optimal doses of burro red blood
cells. This deficiency extended to the production of either 195 or 75
immunoglobulins. These workers also reported depressed'ig.vit£g or
jg_vivg immune responses to aggregated human gamma globulin and
soluble T—independent antigens such as DNP-Ficoll (30). Deficient
responsiveness to sheep red blood cells has also been shown (31-33) as
well as reduced levels of 1961 and IgE antibodies to an unrelated antigen
as measured by passive cutaneous anaphylaxis (34). On the other hand,
investigators have been unable to find depressed antibody responses during
the chronic phase (34, 35).

Specific Antibody Responses. ‘1. cruzi has been described as

 

being immunogenic (10). Evidence has accumulated which indicates the
presence of circulating antibodies directed against the parasite
throughout the course of infection in rabbits, mice, and humans (8,
36—39); these include complement-fixing, hemagglutinating and
precipitating antibodies.

The importance of specific antibodies in resistance to I, Egggi
was first elucidated by Culbertson and Kolodny (40) when they found
increased resistance in mice after passive transfer of anti11.'g[gzi
serum. Kierszenbaum and Howard (41) showed a role for specific antibodies
in their work with genetically-selected lines of mice, Biozzi Ab/L and

Ab/H, differing only in their ability to produce humoral immune responses.

When compared to infected Ab/H mice, 1. Eggzieinfected Ab/L mice had
higher parasitemia and mortality levels and shortened survival times.
Furthermore, mere administration of passive anti11.'gggzi antibodies
eliminated the difference. Krettli and Brener (42) found that bloodstream

forms of the parasite were rapidly agglutinated ig_vitro by sera from

 

chronically infected mice or humans and that the number of trypanosomes
was reduced after such treatment. Circulating forms of I} g:ggi_were
thought to be insensitive to immune lysis for many years. This concept
was revised in 1975 when bloodstream forms of the parasite were described
to be lysed jg_vitrg by specific antibodies from chronic patients or

mice with Chagas' disease (43) and later on confirmed in other
laboratories (44-48). Complement was an absolute requirement for the
lytic reaction and both the classical and alternative pathways of
complement activation provided lytic activity (43).

Cell Mediated Immunity (CMI)

 

Protective effects of CMI. The importance of the cellular arm of

 

the immune system in resistance to I, Egggi became apparent when the

lack of a functional thymus was found to result in marked exacerbation of
the course of the infection (47-49). Administration of anti-thymocyte
serum (47), neonatal thymectomy (48) and use of congenitally athymic mice
were the selected means for disabling thymic function.

CMI has also been shown to be important in controlling the infection
during the later phase of the disease. Thus, when chronic mice were
immunosuppressed with cyclophosphamide (50,51) or by lethal irradiation
(50), parasitemias, which had become undetectable, re-occurred and other

acute symptoms reappeared.

Delayed Type Hypersensitivity (DTH). Two methods have been commonly

 

used to establish the presence of DTH reactions in I. grgziginfected
mice. Namely, skin reactivity to parasite antigen and the jg_yjtrg_
correlate of DTH, the macrophage migration inhibition test (MIF).
Presence of DTH has been shown by both methods in both man and in mice
with chronic Chagas' disease by several investigators (35,37,39,52-56).
The most extensive report concerning humoral and cellular reactivity in
chagasic patients is that by Montufar et 21: (35). They found that
during the chronic phase leukocyte migration was inhibited (as it would
occur in normal individuals). Skin sensitization with non-I:_grgzi
antigens lead to a detectable immune response and the relative levels of
peripheral blood T- and B-lymphocytes were not significantly different
from normal subjects.

Comprehensive studies to evaluate variations in immunological status
from the beginning of the infection have not been feasible with humans
owing to difficulties in dating the origin of the infection. Such studies
can be undertaken with laboratory animals causing infection under
conditions that lead first to an acute and then to a chronic stage. The
results of such an experiment is a subject of a part of this thesis.

Cytotoxicity Mechanisms. Involving Unsensitized Cells. In recent

 

 

years, a considerable amount of work has focused on the ability of
unsensitized cells to lyse parasitic agents in the presence of specific
antibodies. These cytotoxic mechanisms have been referred to as
antibody-independent cell-mediated cytotoxicity or ADCC. Eosinophils,
neutrophils, macrophages, and lymphocytes from rats, mice, and humans have

been shown to have the capability to lyse culture forms (epimastigotes) of

I, grgzi (57-62). The unfortunate drawback of this vast amount of
research was the use of epimastigotes, a form rarely found in the
mammalian host. Kierszenbaum and Hayes (63,64) and Okabe et_gl. (65)
were able to demonstrate that bloodstream forms were also susceptible to
lysis by human and mouse cells via ADCC mechanisms. The former group was
able to show effector activity in this system for eosinophils,
neutrophils, and lymphoid cells of human and mouse origin.

Cytotoxicitngechanisms. Involving Sensitized Cells. The ability

 

 

of sensitized cells to specifically lyse I. grggi_remains a
controversy. Kuhn and Murnane (66) described the specific cytolysis of
parasitized syngeneic fibroblasts jg_yjt:g_by spleen cells from

acutely infected mice. The rate of cytolysis was estimated by assaying
51Cr- release. Normal, uninfected fibroblasts were not affected,
suggesting an immune response directed against parasite antigens expressed
by host cells during the acute phase. On the other hand, Hanson (67)
could not find significant lymphocyte-mediated lysis 0f.I-

cruzi-infected kidney cells or syngeneic macrophages.

Destruction of T. cruzi by Macrophages. Macrophages have been a

 

 

central target for study for many years. In 1959, Taliaferro and Pizzi
(68) demonstrated that these cells were both a target for and an
adversary of the parasite. Thus, epimastigotes were shown to be readily
destroyed within macrophages. More recent studies have demonstrated the
ability of macr0phages to kill virulent blood forms jg yiyg_and jg_
113:9. Kierszenbaum gt 31. (69) injected mice with silica, a

macrophage-killing agent, and infected them with trypomastigotes.

10

Resistance was reduced as measured by mortality rates and parasitemia.
Macrophage stimulation had the opposite effect.

Activated macrophages demonstrate a greater-than-normal capacity to
take up and destroy I. grgzi. Mouse macrophages stimulated 1g

vivo with BCG, Corynebacterium parvum, or other activators of

 

mononuclear phagocytes readily kill the parasites (70-72). Similar
results have been reported for activated human macrophages (73).

Suppression of CMI

 

Altered Responsiveness to Mitogenic Stimulation. Several

 

investigators have reported suppression of responses to mitogens. In
mice immunized with a crude sonicated preparation of I, g:gzj_(CSA)

and infected with bloodstream forms of I. eggzi, lymphocyte

responses to phytohemagglutinin (PHA) were markedly reduced when
compared to mice which received only CSA (74). A similar disparity in
responses to CSA was shown by using a dermal test. Ramos gt 21°

(15) reported decreased responses in acutely infected mice to
concanavalin A (Con A) and bacterial lipopolysaccharide (LPS).
Kierszenbaum and Hayes (51, 75) evaluated lymphocyte responsiveness to
PHA, Con A, and LPS in inbred mice during the different stages of the
disease. Responses to all three mitogens were severely inhibited during
the acute phase but normal responses were seen during the chronic stage.
It was also noted that during the acute period splenic T lymphocytes
were reduced both in absolute number and in percentage. Whereas B
lymphocytes increased in absolute numbers but their percentage varied
within narrow limits. Normalization of the lymphocyte population in the

spleen during the chronic period suggest that immunological events may

ll

play a role in supporting the transition from acute to chronic Chagas'
disease.

Depressed Drug-Induced Contact Sensitivity. In studies of contact

 

sensitivity Reed et 31. (76,77) examined the ability of I.
grgziginfected mice to respond to the sensitizing drug oxazolone.

Mice which had been sensitized prior to infection lost their
responsivess after infection. Spleen cells from infected mice
transferred to normal mice responded to the agent; transfer of
sensitized non-adherent cells to uninfected mice effected responses but
non-adherent cells transferred to infected mice could not increase
responses. In contrast, the transfer of normal adherent cells
significantly improved responsiveness. From these observations, these
investigators concluded that macrophages from infected mice were
altered. The possibility that the macrophages themselves could have
been suppressive was not addressed.

Mechanisms of Immunosuppression

 

Suppressor T Lymphocytes. Ranos g g. (15) reported that

 

spleen cells from infected mice were able to suppress mitogen-induced
responses of normal mice. The cell responsible was claimed to be
non-adherent and removable by treatment with anti-mouse thymocyte serum
plus complement. These results could not be confirmed by several other
studies in which removal of Lyt 2.1-bearing cells -which include the
suppressor T cells- failed to restore responsiveness (12, 76-78).

Adherent Suppressor Cells. Macr0phages have been implicated as

 

mediators of the suppression seen during the acute phase of infection

(76,79). In mitogen-induced mouse spleen cell response assays,

12

Cunningham and Kuhn (74) noted that macrophage-enriched spleen cells
were suppressed during acute infection. Macrophage-depleted spleen
cells had greater responses, though responses did not return to normal.
Kierszenbaum (76) reported that reduced mitogen-induced responses were
seen when purified adherent spleen cells from infected mice were
co-cultured with normal spleen cells. The responses were enhanced by
exchanging the adherent cells for those from uninfected mice. In
addition, treatment of infected spleen cells with indomethacin -a drug
known to block the cyclooxygenase pathway of biosynthesis of
prostaglandin products- improved their responses to T and B
cell-specific mitogens.

Parasite—Induced Suppression. A role for I-.££E£i in directly
modifying cell-mediated responses was initially disqualified by Ramos
.gt_al. (15). In contrast, a more in-depth study of this phenomenon

is presented in Chapters II and III.

10.

11.

12.

13.

14.

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of complement depletion in experimental Chagas' disease. Imnune lysis

of virulent blood forms of Trypanosoma cruzi. Infect. Immun. ll:
86-90 0

 

Krettli, A. U. and R. S. Nussenzweig. 1977. Presence of
immunoglobulins on the surface of circulating trypomastigotes of
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complement and lysis. PAHO Sci. Publ. No. 347: 71-73.

Krettli, A. U., P. W. Carrington, and R. S. Nussenzweig. 1979.
Membrane-bound antibodies of bloodstream Trypanosoma cruzi

in mice: strain differences in susceptibility to complement mediated
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Kierszenbaum, F. 1976. Cross-reactivity of lytic antibodies
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cruzi: effects of anti-lymphocyte serum and neonatal thymectomy
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Kierszenbaum, F. and M.M. Pienkowski. 1979. Thymus-dependent
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Brener, Z. and E. Chiari. 1971. The effects of some immunosup-
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Hayes, M.M. and F. Kierszenbaum. 1981. Experimental Chagas'
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Seah, S. 1970. Delayed hypersensitivity in Trypanosoma cruzi
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treated specific antigen on inhibition of leukocyte migration.
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lysis of Trypanosoma cruzi epimastigotes by eosinophils and
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Sanderson, C.J., A.F. Ldpez, and M.M. Bunn Moreno. 1977. Eosino-
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Abrahamsohn, I.A. and W. Dias de Silva. 1977. Antibody dependent
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Sanderson, C.J., M.M. Bunn Moreno, and A.F. L6pez. 1978.
Antibody-dependent cell mediated cytotoxicity of Trypanosoma
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Parasitology 76:299-307.

 

Olabuenaga, S.E., R.L. Cardoni, E.L. Segura, N. G.

Riera, and M. M. E. de Bracco. 1979. Antibody-dependent
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Sanderson, C. J., and W. De Souza. 1979. A morphologic
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Requirements for cellular destruction of circulating forms

of I, cruzi in human and murine in vitro systems.

Immunology 40: 61-66.

 

Kierszenbaum, F. 1979. Antibody-dependent killing of bloodstream
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18

Kuhn, R. E., and J. E. Murnane. 1977. Trypanosoma cruzi:
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Hanson, W. L. 1977. Immune response and mechanisms of resistance
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Taliaferro, W. H. and T. Pizzi. 1955. Connective tissue
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Kierszenbaum, F., E. Knecht, D. B. Budzko, and M. C. Pizzimenti.
1974. Phagocytosis: A defense mechanism against infection with
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Kierszenbaum, F. 1975. Enhancement of resistance and suppression
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infection by Corynebacterium parvum. Infect. Immun. 12:
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Exp. ParasTtol. 41:385.

Hoff, R. 1975. Killing in vitro of Trypanosoma cruzi by
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Hyg. 29: 708-710.

 

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sensitivity responses in mice infected with Trypanosoma cruzi.
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transformation as a measure of immune competence during

experimental Chagas' disease. J. Parasitol. 66: 390-398.

19

79. Kierszenbaum, F. 1981. On evasion of Trypanosoma cruzi
from the host immune response. LymphOproliferative responses
to trypanosomal antigens during acute and chronic experimental
Chagas' disease. Immunology. 44: 641-648.

 

80. Kierszenbaum, F. and D. B. Budzko. 1982. Trypanosoma cruzi:
deficient lymphocyte reactivity during experimental acute Chagas'
disease in the absence of suppressor T cells. Parasite Immunol.
4: 441-451.

 

81. Kierszenbaum, F. 1982. Immunological deficiency during
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J. Inmunol. 129: 2202-2205.

 

20

CHAPTER I

VARIATIONS IN CELL-MEDIATED IMMUNITY TO TRYPANOSOMA CRUZI

 

DURING EXPERIMENTAL CHAGAS' DISEASE

ABSTRACT

Mice infected with bloodstream forms of Trypanosoma cruzi

 

were found unable to respond significantly to subcutaneous stimulation
with parasite antigens with a delayed-type hypersensitivity skin
reaction during the acute phase of the disease. Consistent with this
observation, peritoneal cells collected from infected mice during the
acute period were unresponsive in jg liEEQ assays for production

of macrophage migration inhibitory factor. By contrast, both specific
skin reactivity and significant inhibition of macrophage migration
were noted during the chronic stage, i.e., when parasitemias were on
the decline or undetectable and mortality no longer occurred. Given
the similarity between the kinetic changes in cell-mediated immunity
monitored by lg 1119 tests and their jn_yit£g correlate, the

latter was used to explore the possible role of suppressor T cells in
the causation of immunodeficiency recorded during the acute phase.
Peritoneal cells from acutely infected animals failed to alter
production of macrophage migration inhibitory factor by chronically
infected mouse cells when present in mixtures at proportions
representing up to 75% of the total number of cells. Furthermore, the
dilution effect of cell addition on the jg_yjtrg_reaction to the
trypanosomal antigens was comparable whether normal or acutely
infected mouse cells were mixed with those from chronically infected
animals. Removal of Lyt 2.1 bearing cells from suspensions of acutely
infected mouse cells did not restore lymphocyte responsiveness to the
I, £3251 antigens in the macrophage migration inhibition assay.

These results show that the impairment of specific cell-mediated
21

22

immunity of mice infected with I. cruzi is circumscribed to the
acute phase of the disease and do not support a role for suppressor T
lymphocytes in the lack of host responsiveness to the parasite's

antigens.

INTRODUCTION

The deficient status of lymphocytes from mice infected with

Trypanosoma cruzi has been documented in terms of altered

 

reactivity to mitogenic stimulation (1-3), impaired ability to mount
antibody responses to non-trypanosomal antigens jg_yjt:g_(4-6)

and by depressed drug-induced contact sensitivity (7). More recently,
these observations have been extended to specific jg_yit:g
lymphoproliferative responses triggered by I-.££!£i antigens

derived from host (bloodstream) forms of the parasite (8). However,
the available evidence for the deficient immunological status has been
reported to occur ig_yjt:g during the chronic period of Chagas'
disease both in man (9-13) and in mice (8). Furthermore, exacerbation
of the disease in chronically infected mice following
immunosuppressive treatment with cyc10phosphamide (14,15), suggests an
important role of the immune system in controlling the course of 1.
$5251 infection. On this basis, the study of variations in the
specific immunological status of the host assumes importance in
understanding better the delicate balance of host-I..grggi
interaction. In this work, we have examined the pattern of
cell-mediated immune reactivity of mice to trypanosomal antigens
during the course of an experimental infection produced in inbred mice
which first leads to an acute phase and then to the chronic stage of

Chagas' disease.

23

MATERIALS AND METHODS

Four-week-old inbred, female CBA/J mice used in this work were
purchased from the Jackson Laboratory (Bar Harbor, ME).

Bloodstream trypomastigote forms of Tulahuen strain 1, grgzi
were maintained by serial intraperitoneal (ip) passages in mice.
Infected blood was drawn from the retro-orbital venous plexus and
collected in heparinized tubes. Dilutions were made with sterile
RPMI-1640 medium (Flow Laboratories, Rockville, MD). The doses of
.1..g£gzi used for parasite maintenance and for production of

experimental infection were 105

and 25 organisms, respectively, and
were administered ip in a volume of 0.1 ml. Parasite concentrations
were measured by a standardized microscopic method described elsewhere
(16) and expressed as number of I, g:gzi_ml'1. Cultured (mainly
epimastigote) forms were maintained in a biphasic medium consisting of
agar, sheep blood, glucose, liver and brain-heart infusions (17) at
26°C. Epimastigotes used for the preparation of antigenic material
were grown in the same medium except for the absence of sheep blood.
Epimastigotes were harvested during their logarithmic phase of
growth (on day 5 of culture) and washed three times with sterile
phosphate-buffer saline, pH 7.0 (PBS) by centrifugation at 2500 rpm

for 15 minutes at 4°C. The pelleted flagellates were resuspended in

ten volumes of PBS (final concentration of 5 x 108 T, cruzi

24

25

cells ml'l), frozen with a mixture of dry ice-acetone and thawed in

a 37°C water bath. After five cycles of freezing and thawing, the
organisms were disrupted by sonication (for 45-second periods of
exposure at 30 watts) in a Cell Disrupter (Heat Systems, Plainview,
NY). The suspension was maintained in an ice bath during this
treatment. After centrifugation at 35,0009 for 30 minutes at 4°C,

the supernatant was separated, aliquoted and stored under liquid
nitrogen until used. This preparation will be referred to in the text
as the soluble trypanosome antigen (STA).

Infected and uninfected mice were sacrificed at predetermined time
intervals starting on day 5 post infection (p.i.). An aseptic
operation was maintained throughout. Peritoneal lavages with
serum-free Eagle's Minimum Essential Medium (MEM, Flow Laboratories)
were collected from five animals as described by Stuart and co-workers
(18) and pooled. The cells were washed three times with the same
medium and finally resuspended in MEM supplemented with 10% heat
inactivated fetal calf serum (Microbiological Associates,
Walkersville, MD)(MEMS). Cells were counted using a Neubauer

7 viable,

hemacytometer and the concentration was adjusted to 3 x 10
trypan blue-excluding cells ml'1 in MEMS. Capillary tubes (1.4 mm
internal diameter x 100 mm long) were filled with the cell suspension
and plugged at the end with Critoseal (A. H. Thomas, Co.,
Philadelphia, PA). After centrifugation at 700 rpm for eight minutes

at 4°C, the tubes were cut at the cell-fluid interface. The end

containing the cells was placed in a culture chamber (Sterilin,

26

London, England) which was then filled with MEM. Cultures were set up
in duplicate or quadruplicate both in the presence and absence of 100
pl of STA and incubated at 37°C for 24 hr. This amount of STA was
selected because of optimal responses obtained with it in preliminary
tests for production of macrophage migration inhibitory factor (MIF)
performed with cells from chronically infected mice. MIF assays were
also performed with mixtures containing cells from chronically
infected mice and cells from either normal or acutely infected
animals. The composition of the tested cell mixtures will be
described under Results. The sensitivity of the assay was increased
by measuring uniformly enlarged (by projection at constant
magnification) areas of cell migration using a compensating polar
planimeter (Keuffel and Esser model 620005, New York, NY). Results
were expressed in terms of percentage migration inhibition calculated
by the equation % MI = 100(1 - Mi) where Mi is the migration index (Mi
= area of migration in the presence of antigen/area of migration in
the absence of mitogen).

One half ml of cell suspension containing 1.6 x 107 cells in MEM
was mixed with 0.2ml of monoclonal anti-Lyt 2.l antibody (New England
Nuclear, Boston, MA; titer 10'4) diluted 1/30 in MEM and incubated
at 37°C for 15 minutes. The source of complement (C) activity was
guinea pig serum diluted 1/8 in MEM; 0.5 ml of this reagent was added
to the mixture which was incubated further for 30 minutes. The cells
were then washed twice with 50 ml volumes of MEM, counted in the
presence of trypan blue and finally suspended to 3 x 107 viable

cells ml'l.

27

Determinations of footpad reactivity to STA were made on a time
schedule similar to the one followed for the MIF tests but separated
groups of animals were used. Five mice were tested on any given day,
each receiving a subcutaneous injection of 0.05 ml of STA into the
right hind footpad and 0.05 ml PBS into the contralateral footpad.
Footpad thickness was measured with a micrometric caliper 24 hr after
the injections and the difference between the thicknesses of the right
and left footpads calculated. Groups of five non-infected mice
receiving STA and PBS were used to test each batch of STA. No
significant difference in footpad size or swelling was observed in
these animals.

Each set of data presented in the figures of this paper is
typically representative of two or more separate experiments with

identical protocols.

RESULTS

Course of T. cruzi infection in CBA/J mice

 

A typical course of infection produced in CBA/J mice injected with
25 I, grgzj_is depicted in Fig. 1 to serve as reference for the
studies described below. All of the infected mice developed
parasitemias which attained significant levels on days 13-15 pi,
increased thereafter up until days 23-26 pi and then declined until
becoming undetectable. Separate groups of mice were infected to
determine the mortality rate produced by infecting with 25 organisms.
Forty to 60 percent of these animals survived in the various
experiments in which 100-150 mice were used; a representative
mortality curve is shown in Fig. 1. The survivors were considered to
be chronically infected. This criterion is supported not only by the
observations of Laguens et. al. (19) who established histologic and
electrocardiographic similarities between chronic Chagas' in humans
and mice but also by the previous finding that drug-induced
immunosuppression exacerbates the infection in surviving CBA/J mice
whose parasitemias and other signs of the infection have become
undetectable (15).

Cellular reactivity to STA during T. cruzi infection

 

Animals which received STA and PBS during the first 25 days pi
showed no significant differences in footpad swelling (Fig. 2).
Positive reactions denoted by swelling of the footpads injected with
STA were recorded on day 40 and persisted up until day 60 pi when the

determinations were discontinued.

28

29

FIGURE 1

Course of T. cruzi infection in CBA/J mice.
25 trypomastigotes i.p.

l07 T. cruzi/ml blood
o:
l

 

1

IO 20 30
DAYS AFTER

 

Mice received

JL 1

fif 90
INFECTION

8
— °/oCUMULATlVE MORTALITY

PO
(fl

 

O

30

FIGURE 2

Delayed-type hypersensitivity reaction elicited by STA
during the course of experimental T. cruzi infection.
Points represent the average swelling difference between
footpads of five animals. Vertical lines are the standard
deviations. There was no significant difference in thick-
ness between the footpads of uninfected animals receiving
STA and PBS (not shown).

FOOTPAD SWEL LING (mm)

I.OO -

0.75 -

0.50 "

0.25-

“0.25-

 

31

FIGURE 2

I l J

IO 20 3O 4O 50 60
DA YS AFTER INFECTION

32

MIF assays did not reveal significant inhibition of macrophage
migration until days 40-45 pi when a relatively small but reproducible
inhibitory phenomenon was observed in culture chambers containing STA
(Fig. 3). The percentage of inhibition of macrophage migration
followed a trend of continuous ascent with time after this initial
occurrence.

MIF tests were set up using cell mixtures containing varying
portions of leukocytes from mice sacrificed on their 20th and 60th day
of infection. This was done to find out if acutely infected mouse
cell suspensions contained a p0pulation of suppressor cells inhibiting
the production of MIF. Control assays performed with each of the cell
suspensions prior to mixing readily reproduced the capacity and
incapacity of chronically and acutely infected mouse cells,
respectively, to produce MIF following 1g 115:9 stimulation with
STA. However, cell cultures containing 50 or 75% peritoneal cells
from mice sacrificed on day 20 pi failed to significantly alter the
responsiveness of cells derived from animals sacrificed on day 60 pi
(Fig. 4). The effect of dilution of the chronically-infected mouse
cells with cells from either normal or acutely infected mice were
comparable. Similar results were obtained when cells from mice
sacrificed on day 15 pi were used (data not shown).

Using an independent approach we tested responsiveness of acutely
infected mouse cells in the MIF assay before and after removal of the

Lyt 2.1-bearing cells, known to include the suppressor T lymphocyte

33

FIGURE 3

Variations in the % MI during the course of experimental
T. cruzi infection. Solid line, infected mouse cells;
dashed line, normal mouse cells. Points represent the
average of four separate experiments, each of which was
performed in quadruplicate with pooled cells from five
mice. Vertical lines are the standard deviations.

 

°/o MIGRATION INHIBITION

34

FIGURE 3

 

 

l l l l l J

0 IO 20 3O 4O 50 60
DAYS AFTER INFECTION

35

FIGURE 4

Effects of mixing chronically infected mouse cells with
cells from either normal or acutely infected mice on the
% MI. Cells from acutely and chronically infected mice
were obtained on day 20 and 60 p.i., respectively. The
total number of cells in the assay was constant. The
difference between l00% and the percentage of chronic
mouse cells given in the abscissa corresponds to the per-
centage of normal or acute mouse cells present in the
reaction mixture. Points represent the average of dupli-
cate determinations and the vertical lines the standard
deviation. Note that addition of up to 50% cells from
acutely infected mice did not affect the capacity of
chronically infected mouse cells to produce inhibition of
macrophage migration.

MIGRATION INHIBITION

7..

60

4O

20

36

FIGURE 4

 

 

 

F
—— chronic + acute

’ chronic + normal

/
I- /
/
l 1 l J

O 25 50 75 I00

°/oCHRON|CALLY INFECTED MOUSE
CELLS IN MIXTURE

37

subset. As can be seen in Table 1., treatment with monoclonal
anti-Lyt 2.1 antibody plus C had no consequences on the unresponsive
status of the cells. We considered the possibility that cells
responsible for MIF production in our system could have been Lyt 2.1
positive in that their removal might have abrogated MIF production in
the absence of suppressor T cells. For this reason, we carried out
our parallel control experiments with cells from chronically infected
mice and found that their response to the antigen was not
significantly altered after treatment with anti-Lyt 2.1 plus C.

It is noteworthy that the results of the experiments described in
this paper, performed with pools of cells from several animals, were
systematically confirmed when spot checks were conducted by using cells

from individual mice (data not shown).

38

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.cowpumwcwpmoa my xmu co um0w$wcumm wows m soc» mFqu +

.mumuwpaau cw umscowcma mm: ummp
exp cows; to :umm c_ mpcmewcmaxw mpmcmqmm ozu ms» to mmmgm>m wgu mew mupzmmm -

 

 

m H mm o + H.N ass-wpcm wows umpumccw s__muwcocgu
N H Hw ace: Hoovs umpumwcw appmumcoczu
a H moF u + H.N S»S-chm wave uauumccw »_mp=u<
up H FPF wcoc +wuws umpumwcw apmu=u<
cop mcoc move umuuwmcwca
..z.m.m H cowpacmwz & mPFmo.co “caspaace eoce mp_ao

 

 

.pmmp.de mg» :? mpfimo mmzoe umpummcw xp—muwcoccu use
xflmpzum to mmocm>wmcoammx m:p.co,mmp»do;aexp m>wuwmoaifl.m pa; mo Fm>osmc mo pumewm

 

H MAm<H

DISCUSSION

While evidence for impaired nonspecific lymphocyte reactivity during
experimental Chagas' disease has been presented in the literature, these
results show that the immunological deficiency also applies to in vivo
specific responses t0.I-.E£E£i antigens. The impaired expressions
of specific cell-mediated immunity were limited to the early, acute
period of the disease, whereas the chronic phase was characterized by
significant cellular responsiveness to trypanosomal antigens detected
in vivo and in vitro. This kinetic pattern of variation of I.

EEEEI antigen-induced lymphocyte reactivity closely resembled that
previously described for infected mice with respect to the ability of
their spleen cells to mount proliferative responses to stimulation with
T (concanavalin A and phytohaemagglutinin P) or B (endotoxic
lipopolysaccharide) cell-specific mitogens (3,15). It follows from this
correlation that nonspecific polyclonal activators may be used to both
indirectly evaluate the immunologic status of the infected host and
derive information about the course of the disease.

Tests of inhibition of leukocyte migration have proven useful in the
detection of specific cellular reactivity in chronic chagasic patients
before and after chemotherapy (11). In our work, results of MIF assays
performed during the acute and chronic periods of the infection
correlated well with the variations in host reactivity noted in 1g

vivo delayed-type hypersensitivity reactions elicited with STA. This

39

4O

justified the use of the MIF test in subsequent work to explore the
cause of the immunological deficiency. In human Chagas' disease,
chronically-infected patients are those who, remaining infected, survive
the acute phase. We have used in this work a similar criterion by
considering as chronically infected those mice which survived for
extended periods of time. Since approximately 50% of the infected
animals survived and animals sacrificed during the acute period were
randomly chosen, we assumed that one half of these mice would have
survived had they not been used. Lack of responsiveness in the MIF
assay during the acute but not the chronic stage of the infection was a
systematic and consistent finding, suggesting not only that this
phenomenon was characteristic of the acute phase of experimental Chagas'
disease but also independent on the fate of the animals.

Presence of I. 23251 in chronically-infected mice can be readily
demonstrated by the exacerbating effects of immunosuppression with
cyclophosphamide (14,15) evidenced by the reappearance in the
circulation of significant numbers of trypomastigotes and renewed
manifestation of signs of the disease. It is clear, therefore, that
mere presence of the parasite in the host is not the only requirement
for production of the immunodeficient condition. On the other hand,
with injection of 25 parasites relatively high levels of circulating
flagellates were recorded on days 12 through 35 p.i. These should have
constituted an effective antigenic stimulus both in length of time and
dose of stimulation; yet responses to STA were insignificant during this

period of time whether measured in vivo or lg vitro. It is

 

 

41

conceivable that induction of the immunodeficient state may be dependent
on the parasite concentration as suggested by the results of Schmunis
and coworkers (20) who performed MIF tests with cells from outbred mice
infected with different doses and forms of I. EEEEI-

The precise nature of the phenomenon resulting in impaired
immunologic reactivity in experimental Chagas’ disease is not known.
Ramos and coworkers (2) have proposed a role for suppressor T lymphocyte
in the inhibition of mitogen-induced proliferative responses by spleen
cells from mice infected with I. Efifléi- However, presence of up to
50% peritoneal cells from acutely-infected mice failed to cause any
significant diminution of the reactivity of chronically mouse cells in
MIF assays. This observation suggests that suppressor T lymphocytes are
unlikely to be relevant to the inhibition of STA-induced responses. In
keeping with this concept was the observation that removal of Lyt 2.1
bearing cells from suspensions of acutely infected mouse cells had no
consequence on the unresponsive status of these cells to I, £1251
antigens. It should be noted that the use of monoclonal anti-Lyt 2.1
antibody in our work precluded the removal of lymphocytes other than
those bearing the Lyt 2.1 marker. Lyt 2.1-positive cells are known to
include the suppressor T lymphocyte subpopulation in CBA/J mice
(reviewed in 21). Nevertheless, a suppressive role for another type of
regulatory cell is not negated by the present results and we are
currently addressing this possibility. It should be noted that adherent
cells have been proposed as having a suppressive activity in African

trypanosomiasis (22). Also relevant in this context is the recent

42

finding that an immunosuppressive factor present in the sera of mice
infected with I. 21251 requires macrophage-like cells to display its
activity (23). In our own research, the removal of adherent cells from
suspensions of infected mouse spleens has been shown to partially
correct the deficient responsiveness to T and B cell-specific mitogens.
Furthermore, the purified adherent cell population from acutely infected
mouse spleen preparations curtailed the response of uninfected mouse
lymphocytes (24). Although these observations support a suppressor role
for phagocytic cells, whether or not this is the only cause for the
immunological deficiency noted during the acute stage of the disease
remains to be examined.

Recent kinetic studies performed with CBA/J mice revealed a severe
depletion of T lymphocytes in the spleen in the acute phase of the
disease (14). Therefore, there is a possibility that the impaired
immunologic responsiveness of the acutely infected mice may be due, in
part, to a reduction in the number of responder cells.

The present results do not reveal what mechanisms produce the
altered.immunologic status of the infected host and the subject deserves
further attention. Presumably, the parasite benefits from this
condition, evading specific host defenses. A delicate balance between
the capacity of the parasite to induce suppression (directly or
indirectly) and the ability of the immune system to counteract seems to
determine whether or not the infected host will survive, and enter the

chronic stage.

10.

11.

12.

REFERENCES

Rowland, E. C. and R. E. Kuhn. 1978a. Suppression of anamnestic
cellular responses during experimental American trypanosomiasis.
J. Parasitol. 64: 741-742.

Ramos, C., I. Schadtler-Siwon, and L. Ortiz-Ortiz. 1979.
Suppressor cells present in the spleens of Trypanosoma
cruzi-infected mice. J. Inmunol. 122: 1243-1247.

 

Kierszenbaum, F. and M. Hayes. 1980. Mechanisms of resistance
against experimental Trypanosoma cruzi infection.

Requirements for cellular destruction of circulating forms

of T. cruzi in human and murine in vitro systems.

Immunology 40: 61-66.

 

Clinton, B.A., L. Ortiz-Ortiz, W. Garcia, T. Martinez, and R. Capin.
1975. Trypanosoma cruzi: Early immune responses in infected
mice. Exp. Parasitol. 37: 417-125.

 

Ramos, C., E. Lamoyi, M. Feoli, M. Rodriguez, M. Perez, and L. Ortiz-
Ortiz. 1978. Trypanosoma cruzi: Inmunosuppressed response to
different antigens in the infected mouse. Exp. Parasit. 45: 190-199.

 

Rowland, E. C. and R. E. Kuhn. 1978b. Suppression of cellular
responses in mice during Trypanosoma cruzi. Infect. Immun.
20: 393-397.

 

Reed, S. G., C. L. Larsen, and C. A. Speer. 1977. Suppression of
cell-mediated immunity in experimental Chagas' disease. Z.
Parasitenk. 52: 11-17. ‘

Kierszenbaum, F. 1981. On evasion of Trypanosoma cruzi

from the host immune response. Lymphoproliferative responses
to trypanosomal antigens during acute and chronic experimental
Chagas' disease. Immunology. 44: 641-648.

 

Tschudi, E. I., D. F. Anziano, and A. P. Dalmasso. 1972.
Lymphocyte transformation in Chagas' disease. Infect. Immun. 6:
905-908.

Yanovsky, J. and E. Albado. 1972. Humoral and cellular responses
to Trypanosoma cruzi infection. J. Immunol. 109: 1159-1162.

 

Lelchuk, R., A. Patrucco, and J.A. Manni. 1974. Studies of cel-
lular immunity in Chagas' disease: effect of glutaraldehyde-
treated specific antigen on inhibition of leukocyte migration.
J. Inmunol. 112:1578-1581.

Reis, A.P., C.A. Chiari, R. Tanus, and I.M. Andrade. 1976. Cel-
lular immunity to Trypanosoma cruzi infection in mice. Rev.
Inst. Med. Trop. sao Paulo 18:422-489.

 

43

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

44

Montufar, O. M. B., C. C. Musatti, E. Mendes, and N. Mendes. 1977.
Cellular immunity in chronic Chagas' disease. J. Clin. Microbiol.
5: 401-404.

Brener, Z. and E. Chiari. 1971. The effects of some immunosup-
pressive agents in experimental chronic Chagas' disease. Trans.
Roy. Soc. Trop. Med. Hyg. 65:629-636.

Hayes, M.M. and F. Kierszenbaum. 1981. Experimental Chagas'
disease: kinetics of lymphocyte responses and immunological
control of the transition from acute to chronic Trypanosoma
cruzi infection. Infect. Imnun. 31:1117-1124.

 

Kierszenbaum, F. and L. E. Saavedra. 1972. The effects of
bacterial endotoxin on the infection of mice with Trypanosoma
cruzi. J. Protozool. 19: 655-657.

 

Kierszenbaum, F. and D. B. Budzko. 1975. Immunization against
experimental Chagas' disease by using culture forms of
Trypanosoma cruzi killed with a solution of sodium perchlorate.
Infect. Irrmun. 12: 461-465.

 

Stuart, A. E., J. A. Habeshaw, and A. E. Davidson. 1978.

Phagocytes in vitro. lg Handbook of Experimental Immunology.

0. M. Weir, Ed. Vol. 2 Chapt. 1. Blackwell Scientific Publications
Oxford.

Laguens, R. R., P. C. Meckert, M. A. Basombrio, G. J. Chambo, P. M.
Cossio, R. M. Arana, and R. Gelpi. 1980. Infeccion cronica del

raton con Trypanosoma cruzi. Modelo experimental de enfermedad
de Chagas'. Medicina 40: (suppl. 1) 33-39.

 

Schmunis, G.A., H. Vattuone, A. Szarfman, and U.J. Pesce. 1973.
Cell mediated immunity in mice inoculated with epimastigotes

or trypomastigotes of Trypanosoma cruzi. Z. Tropenmed.
Parasitol. 24:81-85.

 

McKenzie, I. F. C. and T. Potter. 1979. Murine lymphocyte surface
antigens. Adv. Inmunol. 27: 338-??.

Wellhausen, S. R. and J. M. Mansfield. 1980. Characteristics of
the splenic suppressor-target cell interaction in experimental
African trypanosomiasis. Cell. Immunol. 54: 414-424.

Cunningham, D. S., R. E. Kuhn, and F. M. Hatcher. 1981.
Trypanosoma cruzi: Effects of infection on cellular response
to alloantigens. Exp. Parasitol. 37: 417-425.

 

Kierszenbaum, F. 1982. Imnunological deficiency during
experimental Chagas' disease (Trypanosoma cruzi infection):
Role of adherent, nonspecific esterase-positive splenic cells.
J. Inmunol. 129: 2202-2205.

 

45

CHAPTER II

SUPPRESSION OF MOUSE LYMPHOCYTE RESPONSES TO MITOGENS I VITRO

BY TRYPANOSOMA CRUZI

 

 

Abstract

The ability of I. .ggggi to inhibit mitogen-induced mouse
lymphocyte responses was studied to find out if the organism itself is
involved in the production of the immunosuppression that occurs during
the acute phase of Chagas' disease. Significant suppression of normal
spleen cell responses to concanavalin A (a T cell-specific mitogen) or
to bacterial lipopolysaccharide (a B cell-specific mitogen) was seen
when the concentration of epimastigote forms of the parasite reached or
exceeded 2.5 x 106 organisms/ml in the cultures. The inhibitory
effect was noted over wide ranges of concentrations of either mitogen.
Since spleen cells stimulated with mitogenic solutions that had been
absorbed with 1 x 107 parasites/ml produced significant responses, the
suppressive effect could not be attributed just to mitogen removal by
the parasites. Preparations of I} grgzi_disrupted by freezing and
thawing also inhibited mitogen-induced responses. This indicated that
production of suppression was not a result of parasite competition for
essential medium nutrients and that trypanosome viability was not
required. Suppression was demonstrable only when the parasites were
incorporated into the cultures within 12 hr after mitogenic stimulation.
These results taken together indicate that I. grgzi has the ability
to modulate directly or indirectly lymphocyte function by interfering

with the initial stages of commitment to lymphoproliferation.

46

INTRODUCTION

As is the case in many other parasitic infections (reviewed by 1),

suppression of immune responses during the acute period of Trypanosoma

 

.grgzi infection (2-7) has been viewed as a biological phenomenon
fostering the establishment and persistence of the flagellate in the host.
Several mechanisms have been postulated for the production of
immunosuppression, including a role for suppressor T lymphocytes (8),
suppressor macrophages (9) and suppressive factors found in the serum of
infected mice (10).. Ramos gt 31. (8) concluded that I. Egggi

could not adversely influence lymphocyte responses because addition of
parasites to mouse spleen cell cultures had no significant consequence on
mitogen-induced responses. At odds with this observation are preliminary
results from our laboratory revealing that marked suppression does occur
when the parasite is incorporated into the cultures. In view of this
difference, we examined in this work the suppressive phenomenon with the
double aim of defining the requirements and kinetics of the effect and

explaining the discrepancy.

MATERIALS AND METHODS

Animals. Five- to six—week-old female CBA/J mice were purchased
from Jackson Laboratory (Bar Harbor, ME).

Parasites. Epimastigote forms were grown in Warren's medium (12)
supplemented with 10% heat-inactivated (56°C, 60 min) newborn calf serum
(KC Biological, Kansas, M0) at 28°C. The cultures were harvested during
the logarithmic phase of growth (day 5) and centrifuged at 800 x g for
10 min. The parasites were then washed 4 times and resuspended at the
appropriate concentrations with medium (see Results). Concentrations of
epimastigotes and trypomastigotes were determined microscopically using a
Neubauer haemacytometer.

Non-living T. cruzi preparations (FTE). Suspensions containing
7

 

 

5 x 10 epimastigotes per ml in medium were subjected to 5 cycles of
freezing (dry ice-ethanol) and thawing (37°C waterbath). No intact
organisms were detectable in any preparation, referred to in the text as
FTE.

Cells. CBA/J mouse spleen cell suspensions were prepared in a
sterile Ten-Broeck tissue grinder (two strokes only) using cold medium.
Tissue debris was removed by filtration through a sterile nylon gauze.
The cells were washed 3 times with medium by centrifugation (240 x g, 10
min, 4°C) and finally adjusted to 1 x 107 viable,
trypan-blue-excluding cells per ml. All cell preparations used in this
study contained >95% viable cells.

Mitogens. Solutions of concanavalin A (Con A, Sigma Chemical Co.,

St. Louis, MO) or bacterial lipopolysaccharide extracted from

48

49

Escherichia coli 055:85 by the phenol-water method (LPS, Difco,

 

Detroit, M1) were prepared in medium and filtered through a sterile
0.45-um pore size filter (Millipore, Bedford, MA). Concentrations of
these mitogens are given in the Results section.

Mitogen-induced DNA synthesis. Cell cultures were set up in

 

triplicate in flat-bottom microculture plates (Limbro, New Haven, CT).
The basic culture system consisted of 0.025 ml of spleen cell suspension,
0.025 ml of medium supplemented with 10% heat-inactivated fetal bovine
serum (Sterile Systems, Logan, UT), 0.025 ml of the appr0priate mitogen
solution and 0.025 ml of medium. When living parasites, or FTE were
incorporated into the culture system, 0.025 ml of the corresponding
preparation substituted for the same volume of medium. The cultures were
incubated at 37°C in a 5% COZ-in-air atmosphere saturated with water
vapor for 72 hr. Each culture received 1 uCi of 3H-thymidine (specific
activity 2 Ci/mmole, New England Nuclear, Boston, MA) in 0.025 ml of
medium 24 hr prior to its termination by processing in a cell harvester.
Radioactivity representing 3H-thymidine incorporation into synthesized
DNA was measured in a liquid scintillation spectrometer.

Cell and parasite viability. Concentrations of intact, motile

 

parasites and trypan-blue-excluding spleen cells were measured

microsc0pically at various times in cultures initiated with 2.5 x 106

6 trypanosomes/ml. The

cells/ml in the presence or absence of 2.5 x 10
final volume of the cultures was 0.1 ml.

Kinetics of parasite effects on spleen cell respgnses to mitogens.

 

Cultures were set up as described under Mitogen-induced DNA synthesis

except for the omission of 0.025 ml medium. After 0, 12, 24, 36 or 48 hr

50

of incubation, experimental cultures received epimastigotes (in 0.025 ml)
so that the concentration would be 2.5 x 106 organisms/ml. Control
cultures received medium instead. Cultures of parasites alone in the
presence or absence of the mitogens as well as mitogen-free spleen cell
cultures were also included as controls. Culture conditions and
processing were also as described above.

Absorption of mitogen solutions with epimastigotes. Solutions

 

containing varying concentrations of Con A or LPS were incubated with 1 x

107

epimastigotes/ml in a 5% C02 incubator at 37°C for 24 hr. The
parasites were then removed by centrifugation (800 x g, 10 min, 20°C)
followed by filtration through a sterile 0.45-um pore size filter. The
supernatants were tested for residual mitogen activity as described above.

Presentation of results. All sets of data presented in the figures

 

and tables of this paper are typically representative of two or more
experiments of identical design. Radioactive counts (cpm) represent the
mean of triplicate determinations‘: standard deviation. Differences
were considered to be significant if P<0.05 as calculated by Student's t

test.

RESULTS

Inhibition of T and B lymphocyte responses by T. cruzi

 

Spleen cell responses to Con A and LPS were markedly inhibited by
epimastigote forms of I. ELEEI when the parasite concentration reached
or exceeded 2.5 x 106 organisms/ml (Fig. 1). Similar suppressive effects
were seen when parasites used at this concentration were co-cultured with
up to 1 x 107 spleen cells/ml, i.e., four times as many cells as present
in the cultures of the experiment depicted in Fig. 1 (data not shown).
This increased number of splenocytes was, however, capable of mounting
significant responses to the mitogens, denoting that culture crowding had
not occurred at these cell concentrations.

T and B lymphocyte responses were inhibited by I, grgzi
epimastigotes over a wide range of mitogen concentrations (Fig. 2 and 3).
The typical supraoptimal zone effect, produced by high concentrations of
Con A, occurred whether or not the parasites were present in the cultures.
However, a shift of peak responses toward higher concentrations of Con A
was often seen when epimastigotes were present. Although LPS-induced
responses are not characterized by a supraoptimal zone effect, suppression
was observed even when the dose of the mitogen was 100 times greater than
the minimal stimulatory concentration. It is noteworthy that actual cell
responses to either Con A or LPS in cultures containing 1, 9:251 had
to be smaller than those depicted in Fig. 2 and 3 because the recorded
values include the contribution of the flagellates.

To find out if reduced responses were due to unusual cell killing in

the presence of T. cruzi, cell concentrations were measured at various

51

52

FIGURE 1

Effect of addition of T. cruzi epimastigotes to spleen

cell cultures on their ability to respond to Con A and LPS.
Dashed curve, responses by cell cultures containing the
indicated concentration of parasites. Solid curve, parasites
alone. Points represent the mean of triplicate determinations
and the vertical bars the standard deviation. The concen-
trations of Con A and LPS were 2.5 and 50 pg/ml, respectively.
Only the differences between the values obtained with egi-
mastigote concentrations equal or greater than 2.5 x 10
organisms/ml and the control value (no parasites present,
ordinate) were statistically significant (P< 0.05), after

the contribution of the parasites was subtracted.

 

 

53

FIGURE 1

ConA

V

b

APB-4

‘
d‘

 

LI’S

--.-1H"

’

I
"P
IT“. 1 11 IM'L” 1 ' lLl I

 

.IO' 5 10.25 50 L" .05

 

JO .5 1.6' 2.5 5.0

MILLION EPIMASTIGOTES I ml

54

FIGURE 2

I

I40

 

 

 

 

 

 

 

120
5’
9 I
K
2
a so
U

40

o .5 ""16' ”'i.s""'£b""é3

Con A ug/ml

Suppressive effect of T. cruzi epimastigotes at varying
concentrations of Con A. Solid curve, spleen cells alone;
dashed curve, spleen cells plus parasites; dotted curve,
parasites alone Cultures contained 2.5 x l05 spleen cells/ml

and/or 2.5 x l06 epimastigotes/ml.

 

55

FIGURE 3

Suppressive effect of T. cruzi epimastigotes at varying
concentrations of LPS. Symbols and conditions are as
described in the legend to Figure 2. Statistically sig-
nificant (P<.0.05) differences existed between the responses
of spleen cell cultures containing and lacking the parasites
at LPS concentrations of 5.0 pg/ml or greater.

I

80

60F

40

l

CPMxio‘3

 

 

56

FIGURE 3

......00.

00.0“- '00..----.--.---000°°.

 

l l .l l l l l

 

 

50I00 200 300 400 500

L P S ug/ml

57

time intervals in cultures containing or lacking the trypanosomes. The
concentration of viable spleen cells declined with time in both cases and
the rates of such decline were comparable (data not shown).

Inhibition of lymphoproliferation is not due to mitogen absorption by T.

 

cruzi

To establish whether inhibition of cell responses was merely due to
mitogen absorption by the parasites, solutions containing the
concentrations of Con A and LPS used in most experiments were tested
before and after incubation with up to 4 times the parasite concentration
used in the experiments described above. The results, presented in Table
1, indicated the occurrence of mitogen absorption in terms of a shift of
maximal responses to Con A towards initially higher mitogen concentrations
and a slight reduction in LPS-induced responses at some of the tested
concentrations. However, significant and even maximal proliferative
responses were readily induced by the absorbed solutions, revealing that
stimulatory concentrations of Con A and LPS had remained after absorption.

Kinetics of T. cruzi-induced inhibition of lymphoproliferation

 

 

Results of experiments in which 2.5 x 105 epimastigotes were added
to cell cultures at various times after mitogen stimulation revealed that
T and B cell responses were significantly suppressed only when the
flagellates were incorporated within the first 12 hr of culture (Fig. 5).
It is of interest that the concentration of epimastigotes, added at time
0, increased threefold over the 3-day incubation period of co-culture with

the spleen cells (data not shown).

58

.cowumw>mu ucmucmpm H mmapm> mpeowpawcp mo cums mg“ mm ummmmgqu mew mu43mmm 4

 

 

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4.4 H 4.5N m.4 H 4.4N o.oo_ was
4., H 4.2N N._ H N.NN o.o~ was
m.o H 4.4. 4.0 H o.F~ o.m was
4.4 H o.~ 4.4 H o.~ o was
F.m H 4.44 4.4 H 4.44 o.m 4 =40
4.N H 4.44 4.4 H 4.NN m.~ 4 coo
m.o H m.m w.m H 4.04 mm._ 4 =48
4.o H 4.4 “.44 H m.m4 m.o 4 =00
o.F H 4.m o.~ H 4.m o 4 =48
:owuqcomne Lmu4<. cowpacomnm mgowmm _E\m:
cowpmgucwucoo
Amuop x Eguv mmmcogmmc ewuavcwucmmopwz :mmopws meuwcH camouwz

 

 

-.mmuom4pmmewam gum: cowquwmam memm new mgommn.mab new < cou.moixpwoeamu 04cmmopwz

H mgm<h

59

.co_umpsewpm uvcwmopwe cmpmm c; N_

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m>4mmmcaasm Amo.o vav “snowmwcmwm >FFmUWHmwumpm .mmcau—zu umumpzswumucmmou4e ow
mmpwmmcma mo cowpwuum Lo mew» mew ucmmmcamc mcmccou we; agave Lona: ms» :4
ummechw was?“ one .30; mEmm ago :4 mecma cmcuo ecu ow mpnmow—gqm Pocpcou

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och .m mczmwd op ucmmmp ms» :4 nonwcommu mm mew mponsxm .mmpomvummswam

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q mmonm

6O

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61

Considering the possibility that inhibited lymphocyte responses could
have been the result of competitive use of essential medium nutrients by
the parasite, experiments were set up in which FTE was substituted for the
viable organisms. As can be seen in Table 2, addition of FTE to the
spleen cell cultures resulted in significant suppression of Con A- and
LPS-induced responses. Two concentrations of each mitogen near optimal
stimulatory levels were systematically used in our experiments. Although
results are shown for one concentration of each mitogen, similar

reductions were observed when the concentrations were doubled.

62

.Amo.o vav “ceo_mwcm4m xppeuwumwumpm we; PmFLmHeE mpwmmgeq mo mucmmnm on» c_
cmmouwe meow mzu sup: umcwepao peep new mape> mwcp cmmzpmn mucmcmemwu as» +

.mecmume w~aco .H mo mucmmnm any :4 :mmoume mgu op mmcogmmc
mcwucoqmmccou we“ op puwnmmc saw: wove—suqu mew: compuaumc 4o mmmepcmugma 4

 

 

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_.m H 4.44 4:02 o.om was
4.44 +4.“ H m.m_ 444 o._ 4 coo
4.0 H 4.44 4:42 o._ 4 :04
4.4 4 4.N 4:42 o 4:42
mha Fe\44
4cowpuzumm 4 top x Ego cowpecmamca cowuecucmucou
m mmcoammm w~sco .h camouwz :mmopwz

 

 

.Amhuv mmpomwpmmewmm szcu .H

mcw>4_coc mo mcowpeceawca mo muom4mm

N m4m<h

m>wmmmcmmzm

DISCUSSION

These results highlight the ability of I, pppgi epimastigotes to
suppress murine T and B lymphoproliferative responses. The effect was
dependent upon the concentration of flagellates and was produced by levels
comparable with the usual parasitaemias occurring during the acute phase
of the disease, i.e., when immunosuppression is seen (6,13). Although
epimastigotes are not found in infected mammalian hosts, the suppressive
effect of this form of I, pppgtias similar to that produced by
bloodstream forms both in magnitude and parasite concentration
requirement (data not shown). Ramos pg 31. (8), did not observe
suppression of mouse spleen cell responses to either Con A or LPS using
parasite concentrations up to 5 x 105 organisms/ml. Since suppression
in our culture system was not seen until the concentration of
epimastigotes reached 2.5 x 106 organisms/ml, the negative results
obtained by these investigators might relate to insufficient numbers of
flagellates in their cultures.

To be noted, the increased DNA synthesis observed in the absence of
mitogen (Figs. 3-5) represented increased parasite multiplication in the
presence of cells and not the reverse (data not shown). Mitogen-induced
responses were diminished despite such increase, indicating that the
extent of suppression of was greater than that represented by the
differences in cpm values.

The present results do not disclose whether living organisms and the
tested preparations of parasite components (FTE) induced suppression by
the same mechanism but do indicate that viability was not a requirement

for production of the phenomenon. Whether parasite components present in

63

64

FTE absorbed Con A could not be ascertained due to inability to separate
soluble parasite material from the mitogen solution after absorption.
Even when mitogen absorption was evidenced by a shift in maximal
stimulatory activity following incubation with epimastigotes, sufficient
activity remained in the solutions to produce Optimal lymphocyte
stimulation. Since the absorptions were performed with four times the
flagellate concentration present in cell cultures it appears unlikely
that the suppressive effect of I, pppgj_resulted merely from mitogen
removal. This demonstration of Con A binding by the parasite is in
keeping with reports by other investigators that Con A agglutinates all
forms of I, gpggi (14-16). Also against a possible role for mitogen
absorption in the production of suppression were the markedly inhibited
lymphocyte responses that were consistently seen when the concentrations
of Con A and LPS were increased to levels well beyond those producing
optimal responses. In fact, a supraoptimal zone effect was observed at
high concentrations of Con A whether or not I. 95251 was present in
the cultures, indicating that excess mitogen was present. If mitogen
absorption by the parasites had been a major factor in causing
suppression, optimal responses would have been recorded before
occurrence of a supraoptimal dose effect but this was not the case.
Although epimastigote growth occurred under our culture conditions,
three observations rendered unlikely the possibility that reduced
lymphocyte responsiveness was due to crowding of the culture by the
parasites. First, in experiments carried out with 1 x 107 cells/ml

-i.e., the same number of cells as the combined number of cells and

65

parasites present in co-cultures at the end of the incubation period-
optimal responses to Con A or LPS were recorded. Second, living and
nonliving (FTE) I. gpggi preparations were equally suppressive.

Third, incorporation of 3H-thymidine by the parasites alone was
relatively small (Fig. 1-5) and was included in the responses mounted by
cultures containing both parasites and spleen cells. These combined
responses were significantly lower than those produced by the cells
alone.

Significant suppression occurred only when I. p:pgi_was added to
the cultures during the initial 12 hr, suggesting that effective
lymphocyte triggering or activation had been impaired. The parasites
appeared to affect mechanisms leading to DNA synthesis and not DNA
synthesis itself since their addition after 12 hr had no consequence on
T or B lymphocyte responses represented by DNA synthesis during the
third day of culture. The rate of decline of splenic cells in cultures
lacking mitogen but containing parasites was comparable to that of
similar cultures to which I, p:pgi_had not been added. Therefore,
reduced responses are unlikely to result from accelerated cell death as
a direct result of the presence of the trypanosomes.

Although the present results support the concept that the parasite
plays an important role in curtailing immune responses in the infected
host, the mechanisms whereby the effect is exerted remain a subject for

further study.

10.

11.

12.

13.

REFERENCES

Mitchell, G. F. 1979. Responses to infection with metazoan and
protozoan parasites in mice. Adv. Imnunol. 28:451-511.

Reed, S. G., C. L. Larson, and C. A. Speer. 1977. Suppression of
cell-mediated immunity in experimental Chagas' disease.
Z. Parasitenk. 52:11-17.

Ramos, C., E. M. Lamoyi, M. Feoli, M. Rodriguez, M. Perez, and
L. Ortiz-Ortiz. 1978. Trypanosoma cruzi: immunosuppressed
response to different antigens in the infected mouse.

Exp. Parasitol. 45:190-199.

 

Rowland, E. C. and R. E. Kuhn. 1978. Suppression of anamnestic
cellular responses during experimental American trypanosomiasis.
J. Parasitol. 64:741-742.

Teixeira, A. R. L., G. Teixeira, V. Macedo, and A. Prata. 1978.
Acquired cell-mediated immunodepression in acute Chagas' disease.
J. Clin. Invest. 62:1132-1141.

Hayes, M. M. and F. Kierszenbaum. 1981. Experimental Chagas'
disease: kinetics of lymphocyte responses and immunological control
of the transition from acute to chronic Ipypanosoma cruzi

infection. Infect. Inmun. 31:1117-1124.

 

Kierszenbaum, F. 1981. On evasion of Trypanosoma cruzi from the
host immune response. Lymphoproliferative responses to
trypanosomal antigens during acute and chronic experimental Chagas'
disease . Inmunology 44:641-648 .

 

Ramos,C.,I. Schadtler-Siwon, and L. Ortiz-Ortiz. 1979. Suppressor
cells present in the spleen of Trypanosoma cruzi-infected mice.
J. Immunol. 122:1243-1247.

 

Kierszenbaum, F. 1982. Innumological deficiency during experimental
Chagas' disease (Trypanosoma cruzi infection): role of adherent,
nonspecific esterase-positive splenic cells. J. Inmunol. 129:
2202-2205.

 

Cunningham, D. S. and R. E. Kuhn. 1980. Trypanosoma cruzi-induced
suppressor substance. I. Cellular involvement and partial
characterization. J. Irrmunol. 124:2122-2129.

 

Budzko, D. B. and F. Kierszenbaum. 1974. Isolation of
Trypanosoma cruzi from blood. J. Parasitol. 60:1037-1038.

 

Warren, L. G. 1960. Metabolism of Schizotrypanum cruzi Chagas. I.
Effects of culture age and substrate concentration on respiratory
rate. J. Parasitol. 46:529-539.

 

Maleckar, J. R. and F. Kierszenbaum. In press. Variations in cell-
mediated immunity to Trypanosoma cruzi during experimental
Chagas' disease. Ann. Trop. Med. Parasitol.

 

66

14.

15.

16.

67

Araujo, F. G., E. G. Handman, and J. S. Remington. 1980. Binding
of lectins to the cell surface of Trypanosoma cruzi. J.
Protozool. 27:397-400.

 

Alves, M. J. M., and W. Colli. l974. Agglutination of
Trypanosoma cruzi by concanavalin A. J. Protozool. 21:575-578.

Pereira, M. E. A., M. A. Loures, F. Villalta, and A. F. B. Andrade.
1980. Lectin receptors as markers for Trypanosoma cruzi.
Developmental stages and a study of the interaction of wheat germ
agglutinin with sialic acid residues on epimastigote cells.

J. Exp. Med. 152:1375-1392.

 

68

CHAPTER III

INHIBITION OF MITOGEN-INDUCED PROLIFERATION OF MOUSE T AND B
LYMPHOCYTES BY BLOODSTREAM FORMS OF TRYPANOSOMA CRUZI

 

ABSTRACT

The role of virulent forms of Trypanosoma cruzi in modulating

 

mitogen-induced lymphocyte responses was investigated in this work.
Bloodstream forms of I. gppgi inhibited normal mouse spleen cell
responses to Con A and LPS in a dose-dependent manner. Reduced
responses were observed over relatively large ranges of concentration of
Con A (SO-fold) and LPS (160-fold). The inhibitory action of the
parasites could not be overcome by increasing the mitogen dose beyond
optimal levels. Furthermore, absorption of mitogen solutions with four
times as many parasites are used in the proliferation assays revealed
that sufficient mitogen activity remained to produce optimal lymphocyte
responses. Therefore, reduced lymphocyte responsiveness was not due to
absorption of mitogen by the parasite. Inhibited responses were also
seen when a sonicated I. gpggi preparation was used, indicating that
parasite viability was not required to produce suppression. Inhibition
of Con A- or LPS-induced responses by the parasites occurred only when
the trypanosomes were incorporated into the system during the first 24
hr of culture. These results show that virulent forms of I. ggggi

can induce suppression of T and B cell responses Ip.yippg and

suggest that the parasite affects lymphocyte commitment to blastogenesis

during the early stages of lymphocyte activation.

69

INTRODUCTION

A marked immunologic deficiency occurs during the acute period of
Chagas' disease or American trypanosomiasis, caused by the unicellular

hemoflagellate Trypanosoma cruzi (1-7). The immunosuppressed status

 

that ensues is believed to benefit the parasite during the period of its
establishment in host tissues. Several possible mechanisms have been
proposed for this immunosuppression. Suppressor T lymphocyte activity
has been described in acutely infected mice (1) but has not been
confirmed in some laboratories (2-5). Depletion of T lymphocytes (3,
6), presence of a soluble suppressive substance in the serum of infected
animals (7), and suppressor macrophage activity (2,8) have also been
reported. The possibility that the parasite itself could somehow alter
lymphocyte reactivity was initially dismissed by the apparent inability
of circulating forms of I; pppgi to affect mitogen-induced responses
jfl_yi£[g(1). However, preliminary results from our laboratory

indicated that the flagellates do induce suppression when present in the
cultures at appropriate concentrations. In view of this new
development, we have re-examined the possibility that virulent
bloodstream (trypomastigote) forms of IIprggI affect proliferative

T and B lymphocyte responses.

70

MATERIALS AND METHODS

Animals. Cultures were set up with cells from 5- to 6-week-old
inbred (female) CBA/J mice (The Jackson Laboratory, Bar Harbor, ME),
whereas in vivo transfers of the parasite were made in 4-week-old
stock C01 mice (Charles River, Wilmington, MA).

Parasites. The Tulahuen strain of I; ELEEi was used.
Bloodstream forms were maintained by serial intraperitoneal (ip)

passages. Two weeks after infection with 1.5 x 105

organisms, mice
were anesthetized with ether and bled by cardiac puncture. The blood
was aseptically collected into heparinized tubes and centrifuged at 200
x G for l0 min at 20°C The supernatant was applied on top of a
Ficoll-Hypaque mixture of density 1.007 (Lymphoprep, Nyegaard, Oslo) and
centrifuged at 400 x G for 45 min at 20°C (9) to separate the
free-swimming flagellates (interface). The recovered trypomastigotes
were washed three times by centrifugation at 800 x G for 10 min with
RPMI I640 medium (GIBCO, Grand Island, NY) supplemented with 100 U/ml
penicillin and 100pg/ml streptomycin (RPMI 1640A) and adjusted to the
appropriate concentrations with the same medium. Parasite
concentrations were measured microsc0pically by using a Neubauer

hemacytometer.

Sonicated T. cruzi ppeparation (STC). Suspensions containing 1
7

 

x 10 purified II_cruzi/ml in RPMI l640A were subjected to four
45-sec sonication pulses while maintained refrigerated in a dry ice
bath. After centrifugation at 800 x G for 15 min at 4°C , the

supernatant was filtered through a 0.45-pm pore-size filter (Millex;

71

72

Millipore Corp., Bedford, MA) and used immediately. When added to cell
cultures, this preparation provided an amount of trypanosomal material
equivalent to 2.5 x 106 organisms/ml of culture. In some experiments
STC was used after dialysis against 500 vol of RPMI 1640A at 4°C.

.lelg. The spleens of CBA/J mice were aseptically removed and
converted to single-cell suspensions in ice-cold RPMI 1640A by using a
Ten Broeck tissue grinder (two strokes only). The preparations were
filtered through a sterile nylon gauze to remove tissue debris, and the
cells were washed three times with RPMI l640A by centrifugation. Cells
were counted by using a hemacytometer and were adjusted to the
appr0priate concentrations of viable trypan blue-excluding nucleated
cells per ml.

Mitogens. Concanavalin A (Con A; Sigma Chemical Co., St. Louis,

MO) and endotoxic lipopolysaccharide (LPS) extracted from Escherichia

 

.ggII 055:85 by the phenol-water method (Difco, Detroit, MI) were used
to stimulate cell cultures. Repeated purchases of Con A and LPS during
the period of this study resulted in the use of different batches.
Solutions of these mitogens were prepared in RPMI l640A and sterilized
by filtration through Millipore filters of 0.45pm pore size.

Mitogen assays. Duplicate or triplicate cultures were set up in

 

flat-bottom microculture wells (Linbro, New Haven, CT). Each culture

5 or 5.0 x 105

consisted of 0.1 ml containing either 2.5 x 10 cells
alone or cells plus varying concentrations of parasites (see Results),
and the appropriate mitogen concentration. All cultures contained 2.5%
heat-inactivated fetal bovine serum (Sterile Systems, Logan, UT). The

cultures were incubated at 37°C in a 5% C02-in-air incubator

73

saturated with water vapor for 72 hr. One pCi of 3H-thymidine

(specific activity 2 Ci/mmol; New England Nuclear, Boston, MA) was added
to each well 24 hr before termination of the cultures. An automated
culture harvester (MASH II, Microbiological Associates, Walkersville, MD)

3H-thymidine

was used to process the cultures for measurement of
incorporation into synthesized DNA in a liquid scintillation counter.
Results were expressed as the mean counts per minute 1 standard
deviation. Control cultures were simultaneously set up to which mitogens
were not added. Additional controls consisted of parasites alone cultured

with and without mitogen.

Kinetic studies. Spleen cell cultures containing 2.5 x 106

 

cells/ml were stimulated with various concentrations of Con A or LPS and
received trypomastigotes to provide a final concentration of 2.5 x 106
organisms/ml at predetermined times after mitogenic stimulation. Cell
cultures were set up at 12 hr intervals but received the parasites at the
same time, i.e., at different times after initiation of the cultures.
Additional cultures were set up at each time interval that received RPMI
1640A instead of the parasite suspension. Cultures of the parasites alone
in the presence or absence of mitogen as well as mitogen-free cell
cultures were also included. After 48 hr the cultures received 1 uCi of

3

H-thymidine and were terminated 24 hr later as described above.

Absorption of mitogen by T. cruzi. Solutions of Con A and LPS

 

whose concentrations were the same as those used in the mitogenic assays
were incubated with 1 x 107 trypomastigotes/ml at 37°C for 24 hr in a
5% C02 incubator. After removal of the parasites by centrifugation, the

supernatants were tested for residual mitogenic activity.

74

Presentation of results and statistics. Sets of data presented in

 

this paper are typically representative of two or more experiments of
identical design. Differences were considered to be statistically

significant if P<0.05 as calculated by Student's "t" test.

RESULTS

Inhibition of mitogen-induced lymphoproliferation by T. cruzi

 

trypomastigotes. Although optimal mitogen-induced responses were mounted

 

by cultures containing up to 1 x l07 spleen cells/ml (data not shown),

2.5 x106

cells/ml was the concentration selected for most subsequent
experiments. When these cultures were stimulated with Optimal
concentrations of Con A (2.5 ug/ml) or LPS (50 pg/ml), addition of

I, pppgi trypomastigotes significantly reduced H-thymidine
incorporation. The minimal concentration of parasites that induced such

an effect was 2.5 x 106

parasites/ml (Figs. 1 and 2). Therefore,
subsequent experiments were performed by incorporating into the culture
system parasites at 2.5 x 106 organisms/ml. This number of trypanosomes
consistently suppressed mitogen responses by cultures containing up to 1.0

x 107

cells/ml, i.e., four times as many cells as present in our

standard culture system (data not shown). In experiments in which the
concentrations of Con A or LPS were varied over wide ranges, the
inhibitory effects of the trypomastigotes were readily reproduced (Figs. 3
and 4). Similar results were obtained when the number of cells and
parasites were doubled, whereas other conditions remained unchanged (data
not shown). Supraoptimal responses were produced with Con A whether the
parasites were present or not. It should be noted that the actual
lymphocyte responses mounted in the presence of I; pppgi would have to

be smaller than those depicted in Figures 2 and 3 because these data

3

represent the combined uptake of H thymidine by cells and parasites.

75

76

FIGURE 1.

Dose dependence of trypomastigote-induced inhibition of
mouse lymphocyte responses to Con A. (-—-) cells plus

parasites ( ----- ) parasites alone. The concentration of
Con A was 2.5 pg/ml.

im/\I“‘

CPMxlO

 

20

I

“

otggagg..- ..- LL

0 .05 .IO .5 to 2.5 5.0
(T. cruzi/ml) Io'6

 

77

FIGURE 2

Dose dependence of trypomastigote-induced inhibition of
mouse lymphocyte responses to LPS. (-——) cells plus parasites;

 

(----) parasites alone. The concentration of LPS was 50 ug/ml.

6C)
040-
S
X
:2 _
a.
c)

2C)-

,/’/\
O ____ ___ ’a'
0 .05 JO .5 l£>235 5C)

( T. cruzi/ml) lo’6

78

FIGURE 3

I. cruzi-induced inhibition of mouse lymphocyte responses

to Con A over a full dose range. (-——-) cells alone; (----)
cells plus parasites; ( ..... ) parasites alone. Differences
between the responses of cells in the presence and absence
of parasites were statistically significant (P 0.05)

for all mitogen concentrations.

c PM it 10‘3

 

79

FIGURE 3

 

 

 

 

 

 

 

 

IOOF
/\
80' I
- I
60b
4o-
’\
//// \
”TF“// ----- IN. \\\
' -. ....... 1.. ------ *.."".* “N‘
I l I l l ‘ :
I 2 3 4 5 I25

Con A no lml

80

FIGURE 4

T

6C)

CPMxno'?’

 

..4

. .....--...-.---....'...-.o.

 

 

() 5 l0 EKJIOO
LF’S uq/nfl

l l l

 

 

200 EKXD 400

I. cruzi-induced inhibition of mouse lymphocyte responses to
LPS over an extended dose range. Symbols and statistics are
as described in the legend to Figure 3.

81

The concentrations and viability of spleen cells measured after 24, 48,
and 72 hr of incubation in cultures containing or lacking I, pppgi
were comparable (data not shown). Therefore, impaired responsiveness
was not likely to be due to production of cell death by the parasites.

Because I. gpggi is known to bind Con A (9-11), the possibility
that reduced cell responsiveness might have resulted from absorption of
mitogen by the parasite was examined. Results presented in Table 1
showed that despite absorption of Con A solutions with 1 x 107
trypomastigotes/ml (i.e., four times the concentration of I, pppgi
present in the cultures), significant mitogenic activity remained to
induce maximal responses. However, maximal responses were produced with
lower dilutions of the absorbed mitogen solution, indicating that a
certain amount of Con A had been removed by the parasites.

Marked reductions in parasite concentration (75 to 85%) were
recorded 24 hr after initiation of the cultures whether spleen cells
were present or not (data not shown). No additional loss of living
organisms was observed thereafter. Considering the possibility that
parasite products might be suppressive and that viability of the
flagellate may not be a requirement for production of suppression, we
tested the effect of addition of STC to spleen cell cultures. Spleen
cells incubated with an amount of STC equivalent to 2.5 x 106
trypomastigotes/ml showed significantly reduced responses to either Con

A of LPS with respect to those of cultures free of STC (Table 2).

Similar results were obtained with dialyzed STC (data not shown).

82

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83

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N m4m<h

84

In experiments designed to study the kinetics of parasite-induced
inhibition of iymphocyte responses to Con A and LPS, significant
decreases were seen only in cultures that received the trypanosomes

within 24 hr of initiation (Fig. 5).

85

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DISCUSSION

The present results demonstrate the inhibitory effect of virulent
bloodstream forms of I, cruzi on mitogen-induced proliferation of
mouse T and B lymphocytes. Ramos and co-workers (1) did not observe
significant reduction in responses to either Con A or LPS when they added

up to 1 x 105

5

organisms to 0.2-ml mouse spleen cell cultures (i.e., 5 x
10 parasites/ml). In our work, the minimal concentration of
trypanosomes that caused a significant reduction in spleen cell responses
to the mitogens was 2.5 x 106 organisms/ml. This minimal effective
concentration of parasites falls within the usual range of parasitemias
seen in acutely infected CBA/J mice (6). This implies that this condition
for occurrence of parasite-induced suppression exists in infected hosts
and may contribute to the severe immunosuppression that is seen during the
acute period of the disease (13-16).

Whether LPS binds to circulating forms 0f.I-.E[E£i is not known,
but the binding of Con A to these forms has been reported (10-12). Two
lines of evidence denied that absorption of Con A to the parasites may
have caused an apparent lack of lymphocyte responsiveness. First,
supraoptimal zones (decreased responses) were produced with the identical
concentration of Con A whether the parasites were present in the cultures
or not, demonstrating the presence of excess mitogen in both systems. If
significant binding of Con A by the parasites had taken place, increasing
responses would have been expected to occur as the concentration of Con A

increased. However, this was not the case (Fig. 3 and Fig. 5, upper left

87

88

panel). With LPS, the parasites inhibited responses even after a 160 fold
increase in mitogen concentration (Fig. 4). It is therefore unlikely that
absorption of LPS to T. cruzi, if occurring at all, was responsible

for the inhibitory effect. Second, the solutions of Con A used in the
mitogenic assays displayed suboptimal and optimal stimulatory activity
even after being absorbed with four times as many parasites as present in
the inhibition assay system. Therefore, stimulatory levels of mitogen
should have been present in the cultures. Crowding of the cell cultures
by the parasites and competition between cells and parasites for essential
nutrients were considered as hypothetical alternatives to a suppressive
mechanism. Crowding by the trypanosomes (2.5 x 106 parasites/ml) seemed
unlikely in view of the optimal responses that were mounted by cultures
containing up to 1 x 107 cells, i.e., twice the total number of cells

and trypanosomes present in the mitogenic assays. Furthermore,
H-thymidine incorporation by the parasites was relatively small (Figs.
1-5) and was included in the responses mounted by cultures containing both
cells and parasites. These combined responses were nevertheless
significantly lower than the mitogenic responses produced by the cells
alone. Also against crowding was the finding that STC, which did not
contain living trypomastigotes, was also suppressive. This observation
ruled out possible competition for nutrients as a cause for the noted
inhibition. Of interest, suppression of responses to sheep erythrocytes
after intravenous injection of a freeze-thaw preparation of I, cruzi

has been seen in mice (17). The suppressive action of STC also suggested
that presence of live trypanosomes may not be a requirement for production

of immunosuppression.

89

I, cruzi induced significant suppression only when added to the
cultures during the initial 24 hr period. This is the time during which
mitogens must be present to commit lymphoid cells to blastogenesis
(18-20). Thus, it is conceivable that the parasites are interfering with
effective lymphocyte triggering or activation. Our results also suggest
that reduced responsiveness is not a consequence of direct interference
with DNA synthesis by the parasites, because flagellates added after 24 hr
remained in the culture until termination but were unable to cause
significant alterations in cell responses to the mitogens.

These results highlight a potentially important role for bloodstream
forms of the parasite or their products in inhibiting lymphocyte
proliferation, which may be relevant to the immunosuppression that ensues

during acute infection with I, cruzi.

10.

ll.

12.

REFERENCES

Ramos, C., I. Schadtler-Siwon, and L. Ortiz-Ortiz. 1979.
Suppressor cells present in the spleens of Trypanosoma
cruzi-infected mice. J. Immunol. 122: 1243-1247.

 

Cunningham, D. S. and R. E. Kuhn. 1980. Lymphoblast
transformation as a measure of immune competence during
experimental Chagas' disease. J. Parasitol. 66: 390-398.

Kierszenbaum, F. 1981. On evasion of Trypanosoma cruzi

from the host immune response. Lymphoproliferative responses
to trypanosomal antigens during acute and chronic experimental
Chagas' disease. Immunology. 44: 641-648.

 

Kierszenbaum, F. and D. B. Budzko. 1982. Trypanosoma cruzi:
deficient lymphocyte reactivity during experimental acute Chagas'
disease in the absence of suppressor T cells. Parasite Immunol.
4: 441-451.

 

Maleckar, J. R. and F. Kierszenbaum. 1983. Variations in cell
mediated immunity to Trypanosoma cruzi during experimental
Chagas' disease. Ann. Trop. Med. Parasitol. In press

 

Hayes, M.M. and F. Kierszenbaum. 1981. Experimental Chagas'
disease: kinetics of lymphocyte responses and immunological
control of the transition from acute to chronic Trypanosoma
cruzi infection. Infect. Immun. 31:1117-1124.

 

Cunningham, D. S., R. E. Kuhn, and E. C. Rowland. 1978.
Suppression of humoral responses during Trypanosoma cruzi
infections in mice. Infect. Immun. 122: 155-160

 

Kierszenbaum, F. 1982. Immunological deficiency during
experimental Chagas' disease (Trypanosoma cruzi infection):
Role of adherent, nonspecific esterase-positive splenic cells.
J. Inmunol. 129: 2202-2205.

 

Budzko, D. B. and F. Kierszenbaum. 1974. Isolation of
Trypanosoma cruzi from blood. J. Parasitol. 60: 1037-??.

 

Alves, M. J. M. and w. Colli. 1974. Agglutination of Trypanosoma

 

cruzi by concanavalin A. J. Protozool. 21: 575-.

Araujo, F. G., E. Handman, and J. S. Remington. 1980. Binding of
lectins to the cell surface of Trypanosoma cruzi. J. Protozool.
27: 397.

 

Pereira, M. E. A., M. A. Loures, F. Villalta, and A. F. B. Andrade.
1980. Lectin receptors as markers for Trypanosoma cruzi.
Developmental stages and a study of the—interaction of wheat germ
agglutinin with sialic acid residues on epimastigote cells. J.
Exp. Med. 152: 1375.

 

90

13.

14.

15.

16.

17.

18.

19.

20.

91

Clinton, B.A., L. Ortiz-Ortiz, N. Garcia, T. Martinez, and R. Capin.
1975. Trypanosoma cruzi: Early immune responses in infected
mice. Exp. Parasitol. 37: 417-125.

 

Reed, S. G., C. L. Larsen, and C. A. Speer. 1977. Suppression of
cell-mediated immunity in experimental Chagas' disease. Z.
Parasitenk. 52: 11-17.

Rowland, E. C. and R. E. Kuhn. 1978. Suppression of anamnestic
cellular responses during experimental American trypanosomiasis.
J. Parasitol. 64: 741-742.

Kierszenbaum, F. and M. M. Hayes. 1980. Evaluation of lymphocyte
responsiveness to polyclonal activators during acute and chronic
experimental Trypanosoma cruzi infection. Am. J. Trop. Med.

Hyg. 29: 708-710.

 

Corsini, A. C. and M. G. Costa. 1981. Immunosuppression in mice
infected with Trypanosoma cruzi (Chagas, 1909). II.
Trypomastigote crude extract (TCE) suppress the humoral response
in mice. Rev. Inst. Med. Trop. sac Paulo 26: 122.

 

Cunningham, B. A., B. Sela, I. Yahara, and G. M. Edelman. 1976.
Structure and activities of lymphocyte mitogens. In_Mitogens

in Immunobiology. J. J. Oppenheim and V. L. Rosenstreich, Eds.
Academic Press, New York. P. 13.

Gunther, G. R., J. L. Wang, and G. M. Edelman. 1974. The kinetics
of cellular commitment during stimulation of lymphocytes by lectins.
J. Cell Biol. 62: 366.

Greaves, M. R., J. J. Owen, and M. C. Raff. 1974. T and B
Lymphocytes. American Elsevier Publisher Co., New York. P. 88.

SUMMARY AND CONCLUSIONS

Inmunosuppression during acute Chagas' disease is both severe and
complex in its expression. Reactivity to all of the immunologic stimuli
tested to-date. Therefore, it has been difficult for researchers to
elucidate the mechanisms that control and protect against the infection.

The first part of this study analyzed the cellular immune responses
for T. cruzi antigen during the course of infection with this
parasite. This work provided a chronological occurrence, duration, and
extent of the immunodeficiency state. Significant differences in
footpad swelling or inhibition of peritoneal macrophage migration were
observed until day 40 post infection. The reappearance of
responsiveness correlated with the disappearance of parasitemias and
mortalities. Effective immune responsiveness was maintained in the
chronic phase of the disease. This report does not support a major role
for suppressor T lymphocytes because a) mixing cells from chronically
infected mice with up to 50% cells from acutely infected mice failed to
alter the responses of chronic mouse spleen cells. Furthermore, removal
of Lyt 2.1-bearing cells did not restore responsiveness to cells from
acutely infected mice.

The second part of this research was concerned with exploring the
possibility that I, 9:351 can alter lymphocyte responses. It was
established that both culture (epimastigote) and virulent bloodstream
(trypomatigote) forms are capable of reducing mouse splenic cell
responses to mitogens. Of particular interest was the initial finding
that production of the suppression was directly linked to parasite

concentration. The minimal concentration necessary to demonstrate

92

93

suppression was well within the levels of parasites present in the
acutely infected animal. Therefore, the conditions for production of
suppression are given in the infected host. It could also be speculated
that these findings may illustrate a possible reason why
immunosuppression is not seen in chronically-infected animals.

Two pieces of evidence indicated that the immunosuppression was not
solely due to mitogen removal by the parasites. First, mitogen
solutions absorbed with either epimastigotes or trypomastigotes were
still stimulatory. Second, the suppression could not be overcome with
concentrations of mitogen far exceeding those necessary to produce
significant responses.

Non-living preparations of both forms of I; grugj_were also
demonstrated to be suppressive. It can be concluded from these findings
that parasite viability is not a requirement for production of
suppression. Secondly, suppression was not merely due to competition
for essential nutrients in the culture media.

Presence of epimastigotes and trypomastigotes was required within
the first 24 hr of incubation of the cultures for suppression to be
seen, suggesting that the action of the parasites is realized during the
early (induction) phase of lymphocyte activation.

Recent work, described in the Appendix, focused on the cell(s) which
are sensitive to the suppressive effect of the trypanosomes. Results
indicated that macrophage-depleted cultures were subject to suppression
induced by the parasite. Addition of increasing numbers of adherent
cells to cultures containing non-adherent cells did not decrease

responsiveness. Adherent cells treated with trypomastigotes were unable

94

to negatively affect non-adherent cell responses. These results, taken
together, indicated that the parasites are able to regulate lymphocyte
function directly and that macrophages are not a requirement for
suppression to be demonstrated. These results do not rule out the
possibility that macrophage are the mediators of parasite-induced

suppression i vivo.

 

In conclusion, the significance of this work is several fold. It is the
first definitive report to describe host CMI to 3.1-.EEEEi antigen during
the course of experimental infection with the parasite. Second, it advanced
the viewpoint that suppressor T lymphocytes are not involved in production of
immunosuppression. Finally, it established a role for the parasite in
directly modulating lymphocyte functions and gave evidence to the cells
involved. Thus, this research gives insight into the understanding of the
mechanisms involved in host:I.'gruzi interactions underlying Chagas'

disease.

APPENDIX I.

Occurrence of immunosuppression during acute Chagas' disease has
been well documented. Several mechanisms have been proposed to clarify
this phenomenon. Ramos and coworkers (1) described the presence of
suppressor T lymphocytes. However, several other studies have not
confirmed their presence (2-5). Adherent cells as mediators of
suppression in this system have been reported some workers (2,6).

Recently our laboratory established a role for Trypanosoma cruzi in

 

modulating lymphocyte function (7,8). Culture forms (7) or virulent
bloodstream forms (8) of I, g:ugj_when incorporated into
mitogen-induced DNA synthesis assays significantly decrease the normal
mouse spleen cell response. In this work we advance the understanding
of this mechanism by examining the involvement of the two major
populations of cells known to interact in the production of immune
responses, non-adherent cells and adherent cells and their possible
roles in parasite-induced immune suppression.

Spleen cell suspensions were prepared from six-week-old CBA/J mice
(Jackson Laboratories, Bar Harbor, Me) as described elsewhere (8) but
with the following modifications. Erythrocytes were removed by density
gradient separation over Ficoll-Hypaque (Lymphoprep, Nyegaard, Oslo).
Bloodstream forms of Tulahuen strain of T, cruzi were isolated from
acutely infected CDl mice at the time of maximal parasitemias and
purified by a two-step method described previously (8). Cell
responsiveness was assayed by measuring the uptake of radiolabeled
thymidine in response to stimulation with either concanavalin A (Con A)

or a bacterial lipopolysaccharide (LPS) (8). Non-adherent cells were

95

96

TABLE 1

The effect of addition of varying numbers of treated or
un-treated adherent cells on the non-adherent cell response
to Con A and LPS in the presence or absence of T. cruzi.

 

 

 

 

TC Mean

 

Mitogen* % AD TC Treatment** cpm x 10'3 % Decrease***
Con A 0 - - 56.9 i 6.7 -
O + - 10.1 i 0.8 82 2
0.1 - - 58.8 i 4.8 -
0.1 + - 9.0 i 0.6 84 7
0.1 - + 53.8 i 4.0 8.5
1.0 - - 68.1 i 6.8 -
1.0 + - 8.2 i 0.3 88 0
1.0 - + 55.8 i 3.0 18.1
10.0 - - 82.6 i 8.2 -
10.0 + - 8.7 i 0.2 89.5
10.0 - + 80.5 i 0.0 2.5
LPS 0 - - 23.8 i 0.2 -
0 + - 4.1 i 0.5 82.8
0.1 - - 22.5 i 1.1 -
0.1 + - 8.4 i 1.4 62 2
0.1 - + 19.9 i 0.7 11.6
1.0. - - 23.6 i 0.4 -
1.0 + - 7.7 i 0.6 67 4
1.0 - + 22.8 i 3.9 3.4
10.0 - - 26.9 i 0.0 -
10.0 + - 7.9 i 0.1 70.6
10.0 - + 25.6 i 0.7 4.8

 

* Con A, 1.0 ug/ml, LPS so ug/ml.

** Adherent cells treated for 1.5 hr with T. cruzi (ratio
of cells to parasite was 5:1).

*** % decrease with respect to untreated cells in the
absence of parasites.

97

purified by three steps of plastic adherence at 37°C. Cells attached
to plastic petri dishes after the first step of adherence were rinsed
with RPMI 1640 medium (GIBCO, Grand Island, NY) and either treated with
parasites (the ratio of parasites to adherent cells was 5:1) or medium
for 1.5 hr at 37°C. I. grugjytreated or medium-treated adherent

cells were harvested, washed, and added at varying concentrations (see
Table 1) to mitogen-stimulated cultures containing 2.5 x 106
non-adherent cells/ml.

As can be seen in Table 1, non-adherent cells produced significantly
lower responses to either Con A or LPS in the presence of parasites than
cultures incubated in the absence of parasites. Increasing levels of
medium-treated adherent cells significantly enhanced the response of
normal non-adherent cells to the mitogens only when I, cruzi_was not
present in the system. The effect of adherent cells which had been
pretreated with I, cruzi_on the response of normal lymphocytes did
not differ significantly from that caused by medium-treated adherent
cells.

These results indicate that bloodstream forms of I. cruzi are
able to modulate lymphocyte responses to mitogens in the absence of
macrophages. This is the first report indicating a direct effect by the
parasites on lymphocytes. These results do not demonstrate or rule out
the possibility that the trypanosomes induce their suppressive effect on

lymphocytes through macrophage modulation.

cruzi. Int. J. Parasitol. In press?

REFERENCES

Ramos, C., I. Schadtler-Siwon, and L. Ortiz-Ortiz. 1979.
Suppressor cells present in the spleens of Trypanosoma
cruzi-infected mice. J. Immunol. 122: 1243-1247.

 

Cunningham, D. S. and R. E. Kuhn. 1980. Lymphoblast
transformation as a measure of immune competence during
experimental Chagas' disease. J. Parasitol. 66: 390-398.

Kierszenbaum, F. 1981. On evasion of Trypanosoma cruzi

from the host immune response. LymphOproliferative responses
to trypanosomal antigens during acute and chronic experimental
Chagas' disease. Immunology 44: 641-648.

 

Kierszenbaum, F. and D. B. Budzko. 1982. Trypanosoma cruzi:
deficient lymphocyte reactivity during experimental acute Chagas'
disease in the absence of suppressor T cells. Parasite Immunol.
4: 441-451.

 

Maleckar, J. R. and F. Kierszenbaum. 1983. Variations in cell
mediated immunity to Trypanosoma cruzi during experimental
Chagas' disease. Ann. Trop. Med. Parasitol. In press

 

Cunningham, D. S., R. E. Kuhn, and E. C. Rowland. 1978.
Suppression of humoral responses during Trypanosoma cruzi
infections in mice. Infect. Immun. 122: 155-160

 

Maleckar, J. R. and F. Kierszenbaum. 1983. Suppression of mouse
lymphocyte responses to mitogens in vitro by Trypanosoma

 

Maleckar, J. R. and F. Kierszenbaum. 1983. Inhibition of
mitogen-induced proliferation of mouse T and B lymphocytes
by bloodstream forms of Trypanosoma cruzi. J. Immunol.
130: 908-911.

 

98

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