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MODULATION 0F MACROPHAGE-TRYPANOSOMA CRUZI
INTERACTION BY LADIOFERRIN

By

Maria De Fatima C. Lima

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

1986

ABSTRACT

MODULATION OF MACROPHAGE-TRYPANOSOMA CRUZI
INTERACTION BY LACTOFERRIN

 

By

Maria De Fatima C. Lima

Chagas' disease is a chronic debilitating illness caused by the

flagellated protozoan Trypanosoma cruzi. This disease currently

 

affects 20 million people in Latin American countries. In its acute
phase Chagas' disease presents intense inflammatory cell infiltrates
around ruptured parasitized cells. Lactoferrin, an iron-binding
protein, is secreted by neutrophils (cells present in acute chagasic
lesions) upon stimulation. Lactoferrin levels increase during
inflammatory conditions. In this work, evidence is presented that
lactoferrin modulates the outcome of the interaction between mouse
peritoneal macrophages or human monocytes and the intracellular
amastigote or bloodstream trypomastigote forms of I;_££ugi, increasing
the ability of the phagocytic cells to bind and kill the parasites.
The latter capacity was found to be dependent on the presence of iron
in the protein. Pretreatment of human monocytes, mouse peritoneal
macrophages and amastigotes with lactoferrin also increased their
mutual association. These phagocytic cells and amastigotes present
receptors for this protein. Pretreatment of macrophages with
lactoferrin and subsequent exposure to agents that bind to the
protein, prior to exposure to parasites, did not abrogate the

lactoferrin effects, whereas complete inhibition of the effect was

Maria De Fatima C. Lima

seen when the blocking agents were present in the medium together with
lactoferrin and cells. The lactoferrin effect was also abrogated when
the parasites were pretreated with lactoferrin, exposed to blocking
agents and then incubated with the phagocytic cells. Macrophages
exposed to lactoferrin killed parasites at a faster rate than cells
not exposed to the protein. This effect appeared to be due to the
production of oxygen metabolites, since parasite killing was inhibited

by scavengers of 02 , H202 and 102. The ability of lactoferrin to
stimulate phagocytic function was also observed when invasive
trypomastigotes were used instead of amastigotes. Some differences,
however, were observed: a) macrophages required a longer lactoferrin
treatment (24 hr) for the effect to be seen (as opposed to 1 hr when
amastigotes were used); b) the mechanism of killing involved primarily
hydrogen peroxide production since only catalase was found to inhibit
the effect. The role of lactoferrin iron ions in the ability of this
glycoprotein to stimulate macrophage functions was studied next.
Apolactoferrin (iron free lactoferrin) or lactoferrin at 20% or 100%
iron saturation increased the uptake of amastigotes by macrophages.
While the latter two preparations were able to stimulate parasite
killing, apolactoferrin has lost this ability. Complete restoration
of the effect on killing was afforded by the addition of ferric and
ferrous ions and partial restoration with zinc ions. Cupric ions were
ineffective. Transferrin, another iron-binding protein, was unable to
increase the uptake or killing of the parasite by macrophages. Iron
and the OH'radical were involved in the lactoferrin mediated killing
effect, since, in the presence of chelators of the former and

scavengers of the latter, killing was inhibited. In another series of

Maria De Fatima C. Lima

experiments, it was found that lactoferrin levels were increased in
the sera of mice infected with l;_grugi. The protein was also found
bound to amastigotes from the spleen. These results demonstrate that
lactoferrin may contribute to the clearance 0f.l;.E£!£i by phagocytic
cells via stimulation of parasite uptake and killing and that

lactoferrin function is dependent upon its iron content.

To Fernando and Rachel with love

To my parents with admiration

ACKNOWLEDGEMENTS

I would like to thank Dr. Felipe Kierszenbaum for his support and
guidance throughout my years at his laboratory. His patience, under-
standing and sense of humor made my task lighter and easier to
complete.

I want to thank the members of my committee, Drs. Jeffrey
Williams, James Bennett, Pamela Fraker and Frank Dazzo for taking the
time to criticize and make suggestions to my work. I would also like
to thank Dr. Frank Dazzo for opening his laboratory to me during my
rotation, and for the many insights and contributions that he gave to
my research, during the course of our extensive conversations, even if
about unrelated subjects.

I want to thank Drs. Julia Wirth, Mark Connelly, Alfred Ayala and
Hugo Molina for enlightening discussions of science and life in
general, and for the trading of many common goods in the laboratory in
the course of our experiments.

The technical assistance and friendship of Mr. James Kidder, Mrs.
Patricia Hoops and Mr. William Morgan will be deeply missed in my days
ahead. With their help at the eleventh hour, many hairy experiments
were conducted in time to finish a deadline.

A special word of thanks is due to Miss Lisa Beltz. We traveled
the same paths, and despaired over the same problems, but Lisa always

had a word of hope and encouragement at the right time. She has my

vi

deepest gratitude and I know I can never pay back all the help she
gave me, especially in the end when I found myself without my family
at my side.

I thank my family, my sister and brother for all the words of
encouragement and praise, and my parents, who even far away, could
transmit such faith and confidence in me that I had no remedy but try
to perform to the best of my abilities.

And to Rachel and Fernando, I can only offer my work as a small
repayment for all the sacrifices they endured during the long time we
were separated. Fernando, I will never forget the unending stimula-
tion you always gave to me. You believed in me at times when I had

ceased to do so. Without you, I wouldn't have made it.

vii

PREFACE

Tropical diseases are the great forgotten maladies of our times.
No matter that in the third world, where 75% of the world's population
lives, they remain one of the major causes of death. This is not the
world to which I came to do research nor is it the world that I
remember when I think of home. For I too am among the privileged,
having had access to adequate shelter, food, and education, which is
more than two-thirds of the people living in my country can expect,
even today. Given my many privileges, it is fitting that I chose for
my studies one of the six diseases singled out by the World Health
Organization Special Programme for Research and Training in Tropical
Diseases, Chagas' disease.

The work which I carried out in this country, using the many
facilities which I now take for granted, is not likely to lead
directly to a cure for this disease, nor has it contributed to
producing a vaccine, the hope of many. My work, however, is a piece
of the puzzle of scientific knowledge necessary to a complete
understanding of a complex biological process. I can only hope that

this piece falls in the right place.

viii

TABLE OF CONTENTS

Page

List of Figures ................................................. xi
List of Tables .................................................. xii
Abbreviations ................................................... xiv
Introduction .................................................... 1
I. Chagas' disease: an overview ............................. 2
II. Factors influencing parasite-host cell interaction ....... 4
III. Role of inflammatory cells in Chagas' disease ............ 6
IV. Mechanisms of phagocyte killing .......................... 9
V. Lactoferrin: general considerations ...................... 12
VI. Lactoferrin: cell binding and its consequences ........... 16

VII. Lactoferrin: its role in production of oxygen
metabOIiteSOOOOOOOO0.0....0.0000000000000000.0.0.0.0000... 17

VIII. ResearCh goals O..0.00.00......0.0.0.000000002000000...... 18

IV. References ............................................... 20
Chapter I.

Lactoferrin Effects on Phagocytic Cell Function I. Increased

Uptake and Killing of an Intracellular Parasite By Murine
Macrophages and Human Monocytes OOOOOOOOOOIIOOOOOOOOOOOOOIOOOOO 32
Abstract 000......00.00.000.000.0000IOOOIOOOOOOOOOOOOOOO0...... 33

IntrOdUCtion OOOCOOOOOOOOOOOOI.0....0.0.0.000...OOOOOOOOOOOOOOO 35

Materials and MethOdS .0....0...0.0.0.0....OOOOOOOOCOOOIOOOOOOO 36

ReSUItS COCOOOOOOOOOOOOOOOOOOO...0.0.0.000...OOOOOIOOOOOOOOOOOO 43

DiSCUSSion 0.00.0.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 70

References .0...0.00000000000000000000000000IOOOOOOOOOOIOOOOOOO 75

ix

Chapter II. Page

Lactoferrin Effects on the Interaction of Blood Forms of
Trypanosoma cruzi with Mononuclear Phagocytes.................. 79

 

Abstract 0.0COOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00... 80

Introduction .................................................. 81
Materials and Methods ......................................... 83
Results ....................................................... 87
Discussion .................................................... 101

References OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 104

Chapter III.
Lactoferrin Effects on Phagocytic Cell Function. Distinct

Roles in Modulation of Macrophage Interaction with a
Parasite(Trypanosoma cruzi) ................................... 109

Abstract ...................................................... 110
Introduction .................................................. 112
Materials and Methods ......................................... 113
Results ....................................................... 117
Discussion .................................................... 132

References COOOOOOOOOOOOOOOOO0..000......OOOOOOOOOOOOOOOOOOOOOO 136

Appendix 0.000000000000...0.0...O000......OOOOOOOOOOOOOOOOOOOOOOO 139

sumary and ConCIUSions OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOO 145

LIST OF FIGURES

Page

Chapter I
Concentration-dependent binding of 125I LF to

];_cruzi amastigotes (left panel) and Scatchard
plot analysis of the data (right panel).................... 52

Chapter II

Internalization of T. cruzi trypomastigotes by
MPM in the presence—(IS or absence (0 ) of

10 ug LF/m] 0.00.00.00.00.0....OOOOOOOOOOIOOOOOO0.0.0.0....

95

Appendix

Demonstration by direct immunofluorescence of
lactoferrin binding to splenic amastigotes in situ......... 143

xi

Table

10

'11

LIST OF TABLES

Chapter I

Enhancing effect of LF on MPM or HBM association

With AMA 00......OOOOOOOOOOOOOOOOOOOOOOO0.0.0.0000...

Effects of pretreatment of MPM or AMA with LE on
their capacity to associate with the untreated
counterpart 0.0.000...0.0000000000000000000.00.0.0...

Presence of receptors for LE on the surface of
MP", HBM and I;'cru2i AMA CO...OOOOOOOOOOOOOOOOOOOOOO

Summary of protocols 1 through 5 ....................

Effects of ac -MM or anti-LE on the enhancing

effect produced by pretreatment of MPM with

LF or by the presence of LF during the MPM-AMA
interaction .........................................

Inhibition by a: - M or anti-LF of the enhancing
effect of LF pretreatment of AMA on MPM-AMA

aSSOCiation OOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOIOOOOOOOOO

Effect of LE on the capacity of MPM to take up
latex beads .0...O...O...000.000.00.000.0.0.0.0000...

Effects of the presence of LF during co-culture
of HBM or MPM with AMA on their capacity to
kil‘ the paraSite 0.0000000000000000000000000.00.0...

Effects of pretreatment of MPM or AMA with
LE on their capacity to kill AMA ....................

Effect of addition to cultures of MPM
containing AMA on parasite killing ..................

Effects of scavengers of intermediates of

oxygen reduction on the LF induced increase
0f AMA kiIIing by MPM 0.00000IOOOOOOOOOOOOOOOOOO0....

xii

Page

44

46

49
54

55

58

60

62

64

66

68

Table Page

Chapter II

1 Minimal pretreatment time of MPM with LF
required for production of enhanced uptake
and killing of blood forms of I; cruzi .............. 88

2 Effects of pretreatment of HBM with LE on their
capacity to take up and kill blood forms of

T. CFUZi .0...0.00.000000.00000IOOOOOOOOOOOOOIOOOO... 90

3 Variations in the parasite contents of MPM
after LF pretreatment for 2 hr ...................... 92

4 Effects of treatment of MPM with LF after
infection with I;_cruzi ............................. 96

5 Effects of the presence of catalase on the
capacity of LF treated MPM to kill blood forms

Oful; CPUZi OOOOOOOOOOOOO0.0.0.0000...00.00.000.00... 99

Chapter III

1 Effects of lactoferrin iron contents on its
capacity to enhance MPM association with and
killing 0f I:-cru2i OOOOOOOOOOOOOO...OOIOOOOOOOOOOOOO 118

2 Effects of iron chelation on the capacity of LE
to enhance MPM association with and killing of

1;.cru1i 0.0.0....OOOOOOOOOOOOOOCOOOOOOOO0.0.0....0.. 120

3 Effects of DTPA and DF on the capacity of LP to
enhance MPM killing of I;_cruzi ..................... 121

4 Relative capacity of ferric, ferrous, zinc and
cupric ions to restore the capacity of Apo LE to
enhance MPM killing of I; cruzi ..................... 124

5 Lack of transferrin effect on the capacity of
MPM to aSSOCiate With or kill AMA OOOOOOOOOOOOOOOOOOO 127

6 Effects of scavengers of hydroxyl radicals
on I;_cruzi AMA killing by LF treated MPM ........... 130

Appendix

1 Increased levels of LF acute chagasic sera .......... 142

xiii

 

Apo-LF
(IEMM
AMA
BSA
DMEM
DF
DTPA
d—FBS
FBS
HBM
KTBA
LF

MEM
ML-15H
MPM
PBS
SOD

ABBREVIATIONS

Iron-free lactoferrin

Alpha methyl mannoside

Amastigote forms of I;_grugi

Bovine serum albumin

Dulbecco's modified Eagle minimum essential medium
Deferoxamine mesylate

Diethylene triamine pentaacetic acid

Dialyzed fetal bovine serum

Fetal bovine serum

Human blood monocyte

aC-ketobutyric acid

Human lactoferrin

Eagle's minimal essential medium

Modified Leibowitz medium containing 2 mg/L of hemin
Mouse peritoneal macrophages

Phosphate buffered saline

Superoxide dismutase

xiv

INTRODUCTION

INTRODUCTION

1. Chagas' disease: an overview

Trypanosoma cruzi is the flagellated protozoan that causes
Chagas' disease, or American trypanosomiasis (1), a chronic debilitat-
ing illness mostly confined to Latin American tropical and subtropical
countries. However, some cases have been reported from temperate
areas of North America (2,3). Recently, the disease, which was
limited to rural areas of the affected countries where the vector,
bugs of the family Reduvididae, is normally encountered, has been
spreading to more populated areas via transmission by blood
transfusion. It has been estimated that up to 20% of blood donors in
nonendemic urban areas are infected with I;_g£ugi_(4). About 65
million people are at risk of I;_££ggi infection (5). Of these, 15 to
20 million are actually infected. Approximately 10% of the infected
individuals, are expected to develop chronic chagasic cardiopathy. In
some areas, chronic Chagas' disease may be responsible for up to 10%
of the deaths among the adult population (6).

The life cycle of the parasite begins when triatomine bugs
deposit feces containing the metacyclic trypomastigote forms of the
parasite on the skin of the host. Man acquires the infection by
rubbing the contaminated feces into broken skin, mucosa, or the lesion
left by the insect bite. The trypomastigotes then gain access to
local cells where they transform intracellularly to amastigote forms

and multiply by binary fission. After extensive multiplication, some

of the parasites differentiate into trypomastigotes and, upon cell
rupture, escape. Released amastigotes, which are thought not to be
infective, are disposed of by phagocytic cells. Trypomastigotes, on
the other hand, can infect neighboring cells or reach the circulation
where they can be ingested by the vector. Once back in the vector,
the trypomastigotes migrate to the anterior portion of the digestive
tract where they transform into epimastigotes, the dividing form in
the insect. In the hindgut, epimastigotes transform into metacyclic
trypomastigotes, the infective stage for mammalian hosts.

The acute phase of Chagas' disease encompasses the clinical signs
found within the first four months of infection in man and can be
diagnosed by the demonstration of parasites in the blood (7). Fever
is an early symptom, and is usually accompanied by malaise and
headaches (8). Systemic alterations include subcutaneous edema,
increase in the volume of the lymph nodes, hepatomegaly, splenomegaly,
and often acute myocarditis with characteristic alterations in the
electrocardiographical pattern (9). Anemia with low levels of iron,
leukocytosis with neutropenia and lymphoCytosis are also present (10).
Clinical symptoms of Chagas' disease are not always evident. The
infection usually takes a more severe course in children and a higher
mortality index is found during early infancy (9). In general,
mortality during the acute disease is low, around 5 to 10% (9).

The large majority of patients lapse into a phase of asymptomatic
infection that can last for years. Some of the subjects present mild
anomalies, such as cardiac conduction disturbances and esophageal and

colonic disperistalsis (10).

Chronic manifestations of the disease are commonly found many
years after the initial infection and include progressive diffuse
fibrosing myocarditis that leads to cardiomegaly, cardiac failure,
arrythmias, and thromboembolic phenomena (10). Once symptomatic, the
course of the disease is grave, with death occurring within a year
(10). In some countries, the digestive system of some patients may
also be compromised, presenting megaesophagus with dilation and loss
of peristaltic movements and megacolon with dilation and obstruction
(11).

On a cellular level, the response of the host to I;_g£ugi is of
an inflammatory nature. Vianna (12) reported that the intact,
parasitized cells were rarely surrounded by inflammatory infiltrates.
However, immediately following the rupture of the parasitized cell, a
focal inflammatory infiltrate was observed in the immediate area (12).
Mononuclear cells predominate in these infiltrates although
polymorphonuclear cells and eosinophils are also present (13). The
first cells to appear in inflammatory foci are polymorphonuclear cells
which sometimes contain phagocytized parasites, followed by an influx
of mononuclear cells, lymphocytes and monocytes (14). The cellular
infiltrate is accompanied by intense vascular dilation, congestion,
edema and sometimes necrosis. In the chronic phase, the intensity of
the infiltrate does not relate to the number of parasites (15). It
consists predominantly of lymphocytes and is accompanied by fibrosis

(15).

II. Factors influencing parasite-host cell interaction
Host-cell binding and penetration by I;_cruzi are vital to assure

the initial infection as well as to perpetuate the parasite in the

host. However, the mechanisms by which the parasite obtains an
intracellular location remain to be elucidated. Treatment of the host
cells with trypsin (16),oC-mannosidase (17), OC-galactosidase (18),
and N-acetyglucosaminidase (19) decreases the ability of the parasite
to bind to these cells. These results suggest that a glycoprotein(s)
might be relevant in the interaction between host cells and parasites.
This point is further supported by the observation that treatment of
the host cells with lectins results in inhibition of parasite
interiorization (20).

With respect to the parasite, ongoing protein synthesis (21), DNA
synthesis (22), glycoprotein synthesis (16) and glycoprotein
processing (23) are necessary for host cell infection, since
inhibitors of these processes inhibit parasite interiorization into
the host cells. The presence of sugar residues on the cell surface of
different stages of I;_££ugi_has been demonstrated by using lectins
(29,25,26).

That sugar residues may be playing a role in the parasite-host
cell interaction has been shown by the fact that N-acetyl glucosamine
pretreatment of the parasite inhibits interiorization of the parasite
into bovine endothelial cells (27). Some lectins have been reported
to exert the opposite effect (28) For example, concanavalin A, wheat
germ agglutinin, and phytohemagglutinin have increased parasite
attachment to and uptake by macrophages (20,28). Moreover, treatment
of the parasite with exoglycosidases markedly enhances their uptake by
phagocytic cells and their ability to infect nonphagocytic cells

(17,18,19).

Several glycoproteins have been isolated from the surface of 1;
$5951 in attempts to pinpoint the Specific ligand(s) involved in host
cell attachment. A 90 kd surface glycoprotein (29) present on all
stages of the parasite and found in sera of chagasic patients (30) has
been shown to induce partial protection in mice against challenge with
trypomastigotes (31). A complex of glycoproteins with an approximate
MW of 85 kd, which is specific for the trypomastigote stage, has been
purified by wheat germ agglutinin affinity chromatography (32).
Antibodies against this complex have been obtained and the IgG
fraction extensively inhibits interiorization of trypomastigotes into
LLC-MK2 cells (33). Recently, a gene fragment coding for an antigenic
determinant of this protein has been cloned (34). The peptide was
shown to consist of three highly conserved 9—amino acid repeats. This
type of repetitiveness resembles the surface peptides of Plasmodium
falciparum (34). The ability of this peptide to induce protection

against challenge by I;_cruzi has not been determined.

III. Role of inflammatory cells in Chagas' disease

Studies have been conducted with inflammatory cells both to
establish their roles in production of host defense and their ability
to interact with I; 55251, Human neutrophils are able to take up and
kill ];_grggl_amastigotes jg_vlt£g (35). The mechanisms of
cytotoxicity of these cells for parasites have been linked to their
ability to produce toxic oxygen metabolites. Human neutrophils can
also destroy antibody-coated epimastigotes, trypomastigotes or
insect-derived metacyclic trypomastigotes extracellularly (35-41) via

toxic oxygen metabolites (42).

Human eosinophils are also able to ingest and destroy amastigotes
(43) and, in the presence of specific antibodies, can destroy
trypomastigotes (39) and insect—derived metacyclic trypomastigotes
(41) via ADCC. Eosinophil cationic proteins play a role both in the
intracellular and extracellular destruction of I;_c§ugj_jg_vit£g
(39,43).

Macrophages play a dual role in Chagas' disease; they can serve
both as a host cell harboring the parasite, contributing to parasite
dissemination in the body, and as effector cells implicated in the
destruction and clearance of parasites from infected tissues. Trypo-
mastigotes can enter macrophages by either phagocytosis (44) or cell
membrane penetration (45). After being phagocytized, parasites are
found initially within phagosomes, to which lysosomes may fuse (44).
After 24 hours, amastigotes are found in the cytoplasm of the macro-
phage (44), suggesting an escape from the phagosomes via their ability
to cross membranes. 0n the other hand, amastigotes can not penetrate
cell membranes and their only means of entry is by phagocytosis.
Unlike trypomastigotes, amastigotes have never been seen outside of
phagocytic vacuoles (46), and, in the vacuoles, they can be killed by
intermediates of oxygen reduction, mainly H202 (46,47).

Several agents have been found to stimulate the uptake and
killing of trypomastigotes by mouse peritoneal macrophages. Human
fibronectin, a cell surface protein which is increased in inflammatory
states, increases the uptake of I;_££ugj_by macrophages (48) and
fibroblasts (49). I;_grugi_possesses receptors for this protein (48),
which could possibly act by bridging the cells, thus increasing their

association. Exposure of macrophages to lymphokines generated by

antigen-stimulated sensitized spleen cells from mice infected with I;_
ggugi_or BCG (50) results in the destruction of internalized
parasites. Trypanocidal activity is also induced in these cells by
incubating them with supernatants of lymphocytes stimulated with
concanavalin A or bacterial lipopolysaccharide (50). Leukotriene B4
and leukotriene C4, products of the metabolism of arachidonic acid,
are produced in increased amounts by inflammatory cells, such as
polymophonuclear leukocytes and macrophages, after stimulation with
phagocytic or chemotactic stimuli (51). These metabolites have been
shown to increase the capacity of macrophages to associate with I;
grugi_and also to stimulate their cytotoxic activity (52,53).
Interferon-3’, the T cell product implicated in macrOphage activation
(54), has also been shown to enhance the capacity of macrophages to
take up and kill trypomastigotes (55). Treatment of trypomastigotes
with specific antibody enhances their uptake by unstimulated
macrophages in vitrg (56), but does not modify their intracellular
fate (44,56). In contrast, specific antibody will enhance parasite
killing by activated macrophages (56).

A similar trypanocidal activity has been seen when human
monocyte-derived macrophages were exposed to I:_gguzirstimulated
peripheral blood lymphocytes from patients with chronic Chagas'
disease (57). Lymphokines generated by stimulating these lymphocytes
with antigens unrelated to the parasite or with concanavalin A were
also active. In contrast, supernatants collected after incubation of
lymphoid cells from normal donors with I; £5251_antigen did not induce

parasitocidal activity (57,58).

IV. Mechanisms of phagocyte toxicity

Microbial killing by phagocytic cells involves a multiplicity of
mechanisms, all of which are set in motion by two cellular events:
degranulation and the respiratory burst. Degranulation is the process
of fusion between the primary phagosomes and the lysosomes (59). The
lysosomes contain enzymes and other agents that participate in the
killing and degradation of ingested microorganisms. Neutrophils
present two major types of intracellular granules (50); 1) the
azurophil or primary granules, that contain a diverse array of
substances, including acid proteases, glycosidases, 5'-nucleotidases,
arylsulfatase, neutral proteases, cationic proteins, myeloperoxidases,
lysozyme and acid mucopolysaccharides, and 2) the specific or
secondary granules, that contain lactoferrin, alkaline phosphatase, a
vitamin-Blz-binding protein, lysozyme and collagenase. This spectrum
of enzymes is enough to degrade many of the diverse lipids,
polysaccharides, and proteins present in microbes.

Macrophages and monocytes can also destroy ingested
microorganisms. Qualitatively, the spectrum of proteolytic enzymes
produced by macrophages shows similarities with that of neutrOphils
although there may be differences in the level of the activity of
certain enzymes (61). MacrOphages can compensate for this deficiency
by increasing protein production upon differentiation, especially when
stimulated (60).

Another mechanism of killing used by phagocytes is the
respiratory burst, a metabolic pathway dormant in resting cells (62)
whose function is to produce a group of highly reactive microbicidal

agents by the partial reduction of oxygen. The first event in this

10

pathway is an increase in oxygen uptake (63) upon stimulation of the
phagocyte. Stimulation of phagocytosis has also been shown to cause
an increase in glucose oxidation via the hexose monophosphate shunt
(63), a metabolic pathway in which glucose is oxidized to carbon
dioxide and a five carbon sugar with NADPH+ serving as electron
acceptor. NADPH oxidase has been implicated as one of the components
of the membrane complex that reduces 02 to 0; , a metabolite
associated with phagocytosis by neutrophils (64). In addition to
NADPH oxidase, cytochrome o559 (65) and ubiquinone (66) are also
believed to be a part of this complex. Nonactivated macrophages have
a weaker respiratory burst than granulocytes, but can be immuno-
logically activated, a phenomenon that increases the affinity of the
NADPH oxidase for NADPH and brings the activity of the enzyme to
levels comparable to that of neutrophils (67).

The first product of the reduction of 02, 02: , is dismutated
spontaneously to H202, a reaction that occurs primarily at low pH.
The reaction may also be catalyzed by superoxide dismutase at pH 7.0
(58)- H202 is a well known toxic agent (69), whose action can be
potentiated by two different mechanisms: peroxidation, leading to the
production of hypohalous acids, and the Fenton reaction, which
generates 0H' and 102. In the first of these mechanisms, H202,
halide, and myeloperoxidase, present in the granules of the
neutrophils, eosinOphils and monocytes, react to form hypohalous acids
of which hypochlorous acid is the most common due to the availability
of C1' in cells. These compounds are very toxic to microorganisms via

halogenation and oxidation of cell surface components (68).

Macrophage granules are devoid of this peroxidase, possibly having

11

lost this enzyme during the process of monocyte differentiation (60).
Macrophages, however, can also acquire it by endocytosis as has been

shown in the killing of eosinophil peroxidase-coated Trypanosoma cruzi

 

and Toxoplasma gondii by resident peritoneal macrophages (70,71).

 

In the Fenton reaction, H202 can form OH' radicals and 102 in the

presence of Fe++ as illustrated below:

+ 1

H202 + Fe++ iFe ++ + 0H“ + 0H° + 02
or, if Fe+++ is present,
ire+++ + o; Fe++ + 02
Fe++ + H202 Fe+++ + OH’ + OH' + 102

The latter is the Haber-Weiss reaction and also requires the presence
of 0;. . Neutrophils, monocytes and macrophages have been shown to
produce 0H“ (68,71,142). Hhether 102 is produced jg_vivg_is not yet
clear, though the killing of Toxoplasma gondii by macrophages (72,73)

 

and by the enzymatic system, xanthine-xanthine oxidase (which produces
these latter metabolites in vitro) (74), has been inhibited by
scavengers of 102.

The toxicity of DH’ is partly due to its ability to react with
membrane lipids by hydrogen atom abstraction, forming a peroxy
radical. This radical can in turn, affect adjacent lipids in the
membrane (75). Hydroxyl radical can also attack DNA and proteins
forming the same peroxy radical in a chain reaction. Recently, it has
been shown that iron is involved in the decomposition of lipid
peroxides to peroxy radicals, thus amplifying the reaction and

contributing to the membrane damage (75).

12

The role of oxygen metabolites in the killing of I; gguzj_by
phagocytic cells is suggested by the parasite's inability to detoxify
these metabolites. Catalase, an enzyme which decomposes H202. is
absent in the parasite (76) and glutathione peroxidase levels are low
(77). A cyanide-sensitive superoxide dismutase has been reported in
I;.££!£l (78) and a cyanide-insensitive but H202 and azide-sensitive
superoxide dismutase has also been found (79). In agreement with the
presence of superoxide dismutases and the lack of an efficient
mechanism to degrade H202, I; £5251_is found to be susceptible to the
toxic effects of H202. Trypomastigotes and amastigotes have been
found to be sensitive to H202 whether enzymatically generated
(35,80,81) or added as a reagent (35). Lymphokine- and IFN-stimulated
macrophages kill I:_gruzi_by mechanisms involving H202 (55,80).
Coating 1;_gguzi_ with eosinophil peroxidase enables macrophages to
kill trypomastigotes in a process inhibitable by both cyanide and
catalase (70). Killing of amastigotes by unstimulated neutrOphils and
macrophages was also shown to be due to H202 (35.45)- I;_££E£i_i5
not the only parasite susceptible to these metabolites since the
mechanism of killing by activated macrophages of two other

intracellular pathogens, Leishmania donovani and Toxoplasma gondii,

 

was also found to be mediated by these compounds (82).

V. Lactoferrin: general considerations

Lactoferrin, present in the specific granules of neutrophils
(83), is an iron-binding glycoprotein with a molecular weight of 76 kd
(84,85). It was first described in milk (86) and is primarily found
in various secretions (87) such as tears, saliva, nasal and bronchial

secretions, urine, seminal fluid, and cervical mucus. Although

13

lactoferrin is found in different types of exudates, two main cell
types produce it: epithelial cells in glandular tissue and myelocytes
of the granulocytic series in the bone marrow (88). Lactoferrin
isolated from a variety of different sources appears to be the same
protein (89,90).

Transferrin, an iron binding molecule found primarily in serum,
has a significant amino acid homology with lactoferrin (91), but these
two proteins do not cross react immunologically (92) and bind to
different receptors on cells (92,93).

In the presence of one mole of bicarbonate, lactoferrin binds two

moles of Fe++

+ per mole of protein (94,95). However, the glycoprotein
has also been found to form complexes with other metals such as
chromium, manganese, cobalt (96), zinc (97), and copper (95).

Lactoferrin binds Fe+++

with an affinity that, at physiological pH, is
260 times greater than that of transferrin (94). The binding of Fe+++
to lactoferrin is stable to low pH (4.0), whereas transferrin releases

its Fe+++

at pH 5.7 (94).

The concentration of lactoferrin in serum ranges from 0.1 to 1.5
pg/ml in normal non-inflammatory states (98,99). It rises at the
onset of inflammation and is increased in the cerebrospinal fluid of
patients with meningitis (100,101) or cerebrovascular insults (102)
and in the plasma of individuals with acute bacterial infections
(103,104). Burn patients also have higher levels of lactoferrin in
their plasma (10-40,p9/ml) (105). In burn patients with complicating
bacterial infections, lactoferrin values are higher than in burn

patients without infections (106). Lactoferrin levels are also

increased in the plasma of cystic fibrosis patients suffering from

14

acute inflammation of the lungs (107) and in rheumatoid states (108).
This has been correlated to an increase in degranulation of

polymorphonuclear cells jg_situ. Patients with Sjogren's syndrome, an

 

autoimmune disease characterized by chronic inflammation of unknown
cause, also have increased levels of lactoferrin in their saliva
(109). This is thought to represent increased synthesis of the
protein by glandular cells since saliva contains only a few
neutrophils.

In the presence of zymosan and Staphylococcus albus (110), latex

 

beads coated with 196 (111), Escherichia coli (112) or Treponema
denticola (113), neutrophils can release up to 90% of their granular
contents of lactoferrin (111). Some investigators have correlated the
increase in lactoferrin concentration in acute inflammatory states
with increased neutrophil turnover or greater numbers of neutrophils
in the blood (114). In keeping with this hypothesis is the observa-
tion that in patients with spontaneous or chemotherapeutically-induced
neutropenia, plasma levels of lactoferrin are lower than normal
(115,116). These levels increase in patients that go into remission
after treatment, and are noticed before concomitant increases in
neutrophil counts are achieved (47).

Unlike transferrin, plasma levels of lactoferrin do not correlate
well with iron storage in the host since there is no correlation
between lactoferrin concentration in iron-deficient subjects and in
those who are iron replete (118). Van Snick et al (110) reported the
involvement of lactoferrin in the hyposideremia of inflammation. They
observed that neutrophils release apolactoferrin upon zymosan-induced

phygocytosis. The released lactoferrin was able to bind iron to

15

saturation, indicating that it had been released from the cell
iron-free. The Fe-lactoferrin was then shown to bind monocytes. .13
‘1119 studies showed that apolactoferrin injected into rats can cause a
marked decrease in the plasma levels of iron. Lactoferrin was found
to accumulate in the reticuloendothelial system. In contrast, when
human apotransferrin was injected into rats, it increased iron levels.
Endotoxin injected into rats decreases the level of iron in the plasma
and simultaneously increases levels of lactoferrin in several organs,
mainly the liver and lungs. Hhen 59Fe citrate is injected 30 minutes
before the endotoxin, the isotope is found to be associated with
lactoferrin extracted from these organs. These findings suggest a
possible mechanism by which iron is accumulated in the
reticuloendothelial system during inflammation. It is possible that
this is the same mechanism by which iron is made unavailable to
microorganisms in the blood, depriving them of this metal essential
for their growth.

A bacteriostatic effect was, in fact, one of the first physio-
logical functions attributed to iron-depleted lactoferrin (119-123).
The antimicrobial ability of apolactoferrin was reversed when an
excess of iron was supplied to the nutritionally-deprived
microorganisms. Apolactoferrin has also been found to be bactericidal

for some species of bacteria: Streptococcus mutans and Vibrio cholera

 

(124), Pseudomonas aeruginosa and Escherichia coli (125), and

 

 

Legionella pneumophila (126). The antimicrobial activity of

 

lactoferrin was verified by the inability of bacteria to grow when
transferred to iron-rich medium (124). Consistent with the protective

effect of lactoferrin are the findings that two patients suffering

16

from recurring bacterial infections had severely reduced levels of

lactoferrin in their neutrophil granules (127,128).

VI. Lactoferrin: cell binding and its consequences

Several cells of the immune system have receptors for lactoferrin
on their surface and the engagement of the ligand in each of the cells
leads to different biological effects. Mouse peritoneal macrophages
have 2x107 receptors/cell with a Ka of 9x105L/mole (92) as determined
by Scatchard plots of the binding of 125I-labeled lactoferrin. The
binding is specific and cannot be inhibited by transferrin, although
the latter protein also binds to these cells (129). Human monocytes
have a slightly higher receptor density (2x108/cell) with a comparable
affinity constant of 3.75 x 105L/mole. The binding is specific and is
inhibited by ethylenediaminotetraacetic acid and mannose (93).

The binding of lactoferrin to monocytes has been described to
increase their cytotoxic activity for tumor cells (130). Both 20%
Fe-saturated and 100% Fe-saturated lactoferrin were effective, whereas
transferrin was not. The binding of lactoferrin to monocytes and
macrophages has a potent inhibitory activity on granulopoiesis in
vit§g_and in lilfl (131). Eight percent Fe-saturated lactoferrin is
inhibitory at 10'13M, whereas 100% Fe-saturated is inhibitory at up to
10‘17M. Lactoferrin acts on an Ia bearing subpopulation of
macrophages responsible for the production of colony stimulating
factor (132). The inhibitory effect is complex since lactoferrin
inhibits release of prostaglandin E2 and acidic isoferritins from
macrophages. These molecules are in themselves inhibitory for
granulopoiesis (133,134,135). Transferrin, in its iron-saturated

form, has no effect on the release of granulopoietic factors from

17

macrophages, although it suppresses the release of granulocyte
macrophage colony stimulating factor from 0KT4+ T lymphocyte
subpopulations (136). However, the relevance of these observations 1Q
3119 has been recently questioned (137,138).

Lactoferrin also binds to the surface of neutrophils following
secretion from the lysosomal granules (139). The binding of lacto-
ferrin to the neutrophil surface promotes their adhesiveness to each
other and to endothelial cells. In one study, neutrophils were found
to contain 1.35x106 receptors/cell with a Ka of 5.2x106L/mole (140),
whereas in another study two kinds of binding sites were demonstrated:
a low capacity and high affinity site (3.9x104/cell with a Ka of
2.2x109L/mole) and a higher capacity and low affinity site
(7.2x104/cell with a Ka of 6x108L/mole) (141). This discrepancy

remains to be explained.

VII. Lactoferrin: its role in production of oxygen metabolites
Lactoferrin has been shown to be involved in the formation of
some reductive oxygen intermediates. It increases 0H“ production by
human neutrophils, neutrophil particulate fractions and the
xanthine-xanthine oxidase enzymatic system (142). This observation
has been confirmed by some laboratories (143,144) and denied by
another (145). Lactoferrin has also been shown to promote red blood
cell lysis by activated neutrophils (146). While agranular
neutrophils were unable to lyse the target cells, they were able to
generate the same amounts of 0; as intact cells. Supplementing
agranular cells with lactoferrin reverted the lytic capacity to a
level comparable with that of intact neurophils, suggesting that

lactoferrin had an effect beyond the production of 0; in the cells

18

(146). On the other hand, lactoferrin without iron or at low level of
iron saturation (0-4%) has been shown to inhibit lipoperoxidation of
bovine brain liposomes initiated by Fe salts. Apotransferrin was
equally effective (147).

The physiological functions of lactoferrin are far from being
completely defined. Because of its ability to tightly bind iron and
its low concentration in serum, this glycoprotein does not appear to
serve as an iron transport protein. Upon binding to receptors in the
membrane of several cell types, it induces different types of cellular
activities that appear to be contradictory. It is evident that much
more must be studied about this protein to ascribe it a Specific role

in biological processes.

VIII. Research goals

A heavy inflammatory cell infiltrate is found in acute chagasic
lesions. These cells are prone to be continuously stimulated jg_§itu_
by several factors, such as by parasites, as they are released from
bursting infected cells, and by products released from the
inflammatory cells themselves as they come into contact with the
parasites. Lactoferrin, a protein secreted by stimulated neutrophils
during inflammation, is one of these products and might therefore
modulate the activities of other cells found in the lesion.

The focus of this work was to define any modulatory effect that
lactoferrin might have on the interaction of phagocytic cells and
mammalian forms of l;_g£ugi and to examine the relevant mechanisms.

The first chapter describes the outcome of jg_vitrg_interaction
between mouse peritoneal macrophages or human monocytes and the

intracellular amastigote forms of Trypanosoma cruzi in the presence of

 

19

lactoferrin. The possible similarities and differences in the fate of
this form and that of bloodstream trypomastigotes were explored in
Chapter 11.

Since lactoferrin is an iron binding molecule, the contribution
of this metal to lactoferrin's effects on macrOphage-parasite
interactions was investigated by selectively removing and adding iron
to lactoferrin preparations. The results of these studies are
presented in Chapter III.

The possibility that lactoferrin might be found ig_vivg_in the
course of I; cruzi infection was studied next, using a mouse model.
These results are presented in the Appendix section.

Closing this thesis, a summary will highlight the conclusions

derived from this work, their significance and perspectives.

10.

11.

12.

20

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32

CHAPTER I

LACTOFERRIN EFFECTS ON PHAGOCYTIC CELL FUNCTION.
INCREASED UPTAKE AND KILLING OF AN INTRACELLULAR PARASITE
BY MURINE MACROPHAGES AND HUMAN MONOCYTES

33

ABSTRACT

Mouse peritoneal macrophages (MPM) or human blood monocytes (HBM)

co-cultured with intracellular (amastigote [AMA]) forms of Trypanosoma

 

gruzi_in the presence of human lactoferrin (LF) took up greater numbers
of organisms than in the absence of LF; the proportion of phagocytes
taking up AMA was also significantly increased. Pretreatment of either
MPM or AMA with LF also enhanced cell-parasite association. By

immunofluorescence, HBM, MPM and AMA were found bind LF. Using
125

6

I-labelled LF, each AMA was determined to have approximately 1 x
10 receptors for LF. The enhancing effect of LF on cell-

parasite association was inhibited when either rabbit anti-LF IgG or at
-methyl mannoside (Gt-MM) was present during incubation of MPM or AMA with
LF or when AMA pretreated with LF were then incubated with either
LF-blocking agent. Hhile these findings seemed to suggest that LF
increased MPM-AMA association by bridging these cells, the LF effect was
not inhibited when MPM pretreated with LF were subsequently incubated with
either oc-MM or anti-LF. Furthermore, LF stimulated phagocytosis as
denoted by'a significant increase in latex particle uptake after LF
treatment of MPM. The intracellular killing capacity of HBM or MPM was
also stimulated by LF and was denoted by increased AMA destruction after
LF treatments. The possibility that LF only appeared to increase the rate
of AMA killing by simply promoting the engulfment of greater numbers of
AMA that would then be destroyed intracellularly seemed unlikely because
untreated MPM that had already taken up untreated AMA killed greater

numbers of AMA when they were subsequently incubated with LF. The results

34

of experiments with scavengers of oxygen reduction intermediates and of
nitroblue tetrazolium reduction tests indicated that H202, 02: and
102 were involved in the killing of AMA by LF-treated MPM. These
results suggest that LF, a glycoprotein secreted by neutrophils in
greater-than-normal amounts during inflammation, may contribute to

macrophage clearance of AMA released from infected host cells.

35

INTRODUCTION

Inflammatory cells have been postulated to play a role in the

clearance of intracellular (amastigote [AMA]) forms of Trypanosoma

 

cruzi, the etiologic agent of Chagas' disease (1, 2). Thus, unelicited
mouse peritoneal macrophages (MPM) (2, 3), human blood monocytes (HBM) (2,
4), human neutrophils (5) and eosinophils (6) have been shown to
internalize T. cruzi AMA and destroy these organisms within their
phagocytic vacuoles (2, 5, 6). However, our understanding of the
mechanisms promoting uptake of microorganisms by inflamatory cells
remains deficient. Recent work has identified fibronectin, a plasma
protein produced by macrophages (7), as a molecule that can mediate and
modulate the uptake of bloodstream forms of T. cruzi and inert particles
by unelicited murine MPM (8). This observation and the well recognized
accumulation of inflammatory cells in acute chagasic tissue lesions
(reviewed in reference 9) have directed our attention towards the possible
role played by proteins secreted by inflammatory cells in phagocytic cell
interactions with mammalian forms of I._grgzi. Lactoferrin (LF), an
iron-binding glycoprotein found in milk, saliva, gastrointestinal fluids
and blood (reviewed in reference 10), is produced and secreted in discrete
amounts by neutrophils (11, 12) -cells which accumulate at inflammatory
chagasic tissue lesions (9)- and its plasma levels increased during
inflanmatory conditions (13). This information, and the observation that
LF increases the natural killer cytotoxicity of adherent monocytes for
tumor cells (14), prompted the present study in which we tested the

ability of LF to influence the early and subsequent stages of MPM or HBM
interaction with T. cruzi AMA.

36
MATERIALS AND METHODS

Animals. Four- to six-week old Crl:CD-1(ICR)BR Swiss mice were
purchased from Charles River Laboratory (Portage, MI).

Parasites. AMA forms of Tulahuén strain T. cruzi were grown
in ML-15HA medium as previously described in detail (15). The AMA
preparations used in this work consisted of 100%.organisms in the AMA form
and contained 99 to 100% viable parasites (i.e., organisms displaying
their typical vibratilein situ motion). The AMA were washed three times
by centrifugation (800 X G,20 min) with Dulbecco's modified minimal
essential medium containing 100 pg streptomycin and 100 IU penicillin per
ml (DMEM) and finally resuspended in the same medium supplemented with 1%
bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO) (DMEM+BSA)
at 1.25 X 107 organisms per ml.

MPM and HBM. Mice sacrificed by exposure to excess ether

 

anesthesia were injected intraperitoneally with 5 ml of sterile DMEM
supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone
Laboratories, Logan, UT) (DMEM+FBS) containing 10 U Heparin/ml. The
methods for collecting and processing the peritoneal cells and for
preparing the resident MPM monolayers have been described (16). The MPM
monolayers consisted of >981 nonspecific esterase-positive cells with
typical macrophage morphology. Adherent MPM were further incubated under
the same conditions (5% 002 in air saturated with water vapor)

overnight. The methods for collecting and purifying HBM and for setting
up the cultures have been described (2). Cultures of these cells

consisted of >991 viable, nonspecific esterase-positive cells with typical

HBM morphology.

37

Biological reagents. Batches of human LF (at 10 to 20% iron
saturation) were purchased from Sigma. The 196 fraction of rabbit
anti-human LF was purchased from Cappel Laboratories (Hest Chester, PA).
Beef erythrocyte superoxide dismutase ($00) was obtained from Miles
Laboratory (Naperville, IL) and beef liver catalase from Sigma. The
concentrations at which these reagents were used are described below with
the corresponding method.

Measurement of AMA uptake by MPM or HBM. The procedure to
measure AMA uptake by MPM has been described (2). Briefly, MPM or HBM
monolayers received 0.2 ml of AMA suspension and 0.1 ml of DMEM+BSA, and
were incubated at 37°C in a 5% C02 incubator for 2 hr. After removing
the free AMA by several washings with DEM, the cultures were fixed with
absolute methanol, stained with Giemsa and monitored microscopically (see
below). When the effect of LF was to be tested, 0.1 ml of the
corresponding LF solution in DMEM+BSA was substituted for the same volume
of DMEM+BSA. In all cases, not less than 200 MPH or HBM were screened,
recording the number of parasites associated with (i.e., bound to or
internalized by) MPM or HBM, the number of MPM or HBM associated with one
or more AMA and the number of MPM or HBM not associated with AMA. These
values were used to calculate the percentage of MPM associated with AMA
and the average number of T. cruzi per 100 MPM or HBM. Each
experimental and control condition was tested in triplicate.

Effects of anti-LF IgG and ot-methyl mannoside (or-MM). Rabbit
anti-LF IgG and ix-MM (Sigma), which bind LF, were used in five types of
protocols. Protocol No. 1: 0.1 ml anti-LE 196 (final concentration in

the culture fluid 3.3 mg IgGAml) or 0.1 ml oc-MM solution (final

38

concentration in the culture 3 0.25M), 0.1 ml LF solution in DMEM+BSA (10
u9flml), and 0.1 ml of AMA suspension were added in immediate succession

to MPM cultures and incubated for 2 hr (5! C02). Protocol No. 2: MPM

 

monolayers were incubated with 10 ug LF/ml for 30 min, washed with DIEM
and then incubated with anti-LE or oc-MM (at the same concentrations as
above) for 30 min. After washing with DMEM, the cultures received the AMA

and were incubated for 2 hr. Protocol No. 3: MPM monolayers were

 

simultaneously pretreated with 10 ug LF/ml and anti-LF or oc-MM (at the
same concentrations as above) for 30 min, washed with DMEM and then

incubated with the AMA for 2 hr. Protocol No. 4: AMA were preincubated

 

with LE (10 ug/ml) at 37°C for 30 min, washed and then incubated with
anti-LF or oc-MM for 30 min. After washing with DMEM, these organisms

were added to untreated MPM cultures and incubated for 2 hr. Protocol
Ng;_§: AMA were simultaneously preincubated with LE (10 ugflml) and
anti-LF or oeMM at 37°C for 30 min, washed and, after added to

untreated MPM cultures, incubated for 2 hr. In all cases, control tests
were included in the experiments in which DNEM+BSA was substituted for the
tested reagents and normal rabbit IgG was used instead of anti-LF 196.

All conditions were tested in triplicate and the cultures were terminated
and processed as described in the preceding paragraph.

Pretreatment of MPM or AMA with LF. To study the effects of
pre-incubation of either MPM (5% C02 incubator) or AMA (water bath) with
LE on their association with the untreated counterpart, monolayers of the
former and suspensions of the latter were incubated with 10 pg LF/ml in
DMEM+BSA at 37°C for varying periods of time (see Results). After

.several washings with DMEM, co-cultures of the LF-treated cell with the

39

untreated counterpart were set up as described above and in the following
section. For mock treatments, DMEM+BSA was substituted for the LF
solution.

Killing of AMA by MPM or HBM. The co-cultures of MPM or HBM with

 

AMA were set up and incubated for 2 hr as described under Measurement of

 

AMA uptake by MPM. After removing the free AMA, 0.3 ml of fresh DMEM+FBS

 

was added and the MPM monolayers were further incubated for 4 hr. These
cultures were terminated and processed for counting as described above.
Triplicate assays were set up for each experimental and control condition.
LF treatment of AMA-containing MPM. After incubating MPM cultures
with AMA for 2 hr as described under Measurement of AMA uptake by MPM,
the free organisms were removed by washing and 0.3inl of DMEM+BSA alone or
containing 10 ug LF/ml was added. These cultures were then incubated
for varying periods of time (see Results) and washed with the DMEM.
After receiving 0.3 ml DMEM+FBS, the cultures were incubated further until
4 hr had elapsed from the time the free AMA were removed. The cultures
were then terminated as described above. In some,experiments, culture
supernatants were collected at the end of the 4-hr incubation period,
centrifuged, decanted and the fluid at the bottom of the tubes was
screened microscopically for the presence of free AMA.
Effect of scavengers of oxygen reduction metabolites on AMA killing

by_MEM, The protocol described under Killing of AMA by MPM was

 

modified so that different scavengers of oxygen reduction intermediates
could be incorporated into the system. One tenth of an ml of solutions of

recrystalized beef liver catalase (to provide a final concentration in the

culture of 160,000 U/ml), $00 (3,000 U/ml), histidine (10 mM) or sodium

4O

benzoate (10 nM) in DIEMi-FBS and 0.2 ml DKM+FBS were added to MPM
cultures and incubated for 3 hr.After washing with DIEM, 0.3 ml of LF
solution at 10 ug/ml DNEM+BSA (containing the scavenger to be tested at
the concentrations mentioned above) was added and the cultures were
further incubated for 1 hr. The culture fluids were then removed and
replaced with 0.2 ml of AMA suspension plus 0.1 ml of DMEM+BSA containing
the corresponding scavenger (to attain the same final concentrations).
Cells and parasites were co-cultured for 2-hr. Control cultures were
included which were treated with DMEM+BSA instead of LF solution. For
heat inactivation, catalase and $00 solutions were heated at 100°C for
20 min. Six identical cultures were set up for each condition; three of
these were terminated by washing and fixation (see above) at the end of
the 2-hr co-culture period. The remainder were washed and, after
receiving 0.3 ml of DMEM+FBS containing the scavenger, were incubated,
further for 4 hr and then terminated. All cultures were monitored as
described above.

Latex bead uptake by LF-treated and mock-treated MPM. Cultures of
MPM were either pretreated with LE (10 ug/ml) for 30 min before receiving
0.2 ml of latex bead suspension containing 2.5 X 106 beads/ml (average
diameter = 1m, Sigma) and 0.1 ml DMEM+BSA or received the LF and the
latex beads at the same time. Control cultures did not contain LF. The
amount of latex beads used in these tests was smaller than usually used in
this type of test because we wanted to use as many particles as AMA were
used in the AMA uptake assay. After incubating the cultures for 2 hr (5%
C02), the monolayers were washed, fixed and stained with Giemsa. The

percentage of latex-containing MPM and the number of latex beads per 100

41

MPH were calculated from parameters similar to those measured in MPM-AMA
interaction experiments.

Nitroblue tetrazolium test. MPM were incubated with DMEM+BSA alone

 

or containing 10 ug/ LF/ml at 37°C for 1 hr, washed and incubated with
AMA in the presence of nitroblue tetrazolium at 0.5 mng for 2 hr. After
washing with DIEM, the cultures received 0.3 ml fresh DiEMi-FBS and, after
another 30 min incubation period, they were fixed with absolute methanol
and tested for the presence of insoluble blue/black formazan as described
by Murray and Cohn (17).

Immunofluorescence tests for LF binding to AMA, HBM or MPM. AMA,
HBM or MPM fixed with 0.25% formaldehyde were incubated (37°C, 30 min)
with DMEM+BSA alone or containing 10 or 100 ug LF/ml. After several
washings, these preparations were incubated (37°C, 30 min) with
heat-inactivated normal rabbit serum, washed and then incubated (37°C, 30
min) with a solution of fluoresceinated anti-LF 196 (10 mg IgGAnl,
Cappel). The slides were then washed, air dried and examined with a
fluorescence microscope.

1251 labeling of LF and determination of receptors for LF on AMA.
One half ml LF solution (at 3.1 mg/ml in phosphate-buffered saline

1251 (specific

solution, pH 7.0, PBS) was mixed with 50 ul of Na
activity 15 mCi/ug I; Amersham Corp., Arlington Heights, IL) in a vial
containing 100 ug Iodogen (Pierce Chemical C0., Rockford, IL) and
incubated at room temperature for 15 min. Unbound radioactivity was
removed by gel filtration through Sephadex G-25 (Pharmacia, Piscataway,

NJ) equilibrated with PBS and the radiolabeled LF was concentrated by

ultrafiltration through collodion bags (Schleicher and Schuell, Keene, NH)

42

The material used in the experiment depicted in Fig. 1 had a specific
activity of 8.74 x 105 cpm/Ug LF. The number of receptors for LF

present on the surface of T. cruzi AMA was determined as follows: in
1.5-ml conical tubes (Bio-Rad, Richmond, CA; catalog 223-9501) pre-coated
with a 20% solution of BSA in PBS, 100-ul aliquots of AMA suspension (1.2
X 107 organisms/ml in Hank's balanced salt solution containing 1% BSA)

were mixed with an equal volume of PBS containing increasing amounts of
125

125

I-labelled LF. After incubation at 37°C for 1 hr, the non-bound

125

I-LF was removed by centrifugation and two washings. Bound I-LF

was determined by measuring radioactivity in the pellet with a gamma

125I-LF was calculated by subtracting the amount bound

counter and free
from the total amount added. In competition type of experiments using
cold LF the assays were conducted as above except that an additional 100

pl of LF or human hemoglobin (Sigma) solution (both at 3.3 mg/ml in PBS)
125

was added. In these experiments, only one concentration of I-LF was
used: 250 ug/ml.

Presentation of results and statistical analysis. All results
presented in this paper are expressed as mean :_SD. Differences were
considered to be statistically significant if P<0.05, calculated by the

Mann-Hhitney “U“ test.

43
RESULTS

Lack of effect of reagents on the viability of AMA, MPM or HBM.
None of the reagents used in this work, including LF, affected the
viability of MPM or HBM (determined by trypan blue exclusion) when
incubated with these cells for up to 9 hr or the viability of T. cruzi
AMA (evidenced by their typical vibrational motion in situ when
incubated with the parasites for 6 hr under the same conditions as
selected for our experiments.

Effects of LF on the interaction of AMA with MPM Presence of LF in
co-cultures of MPM or HBM and AMA during the 2-hr incubation period
increased cell-parasite association as evidenced by significant elevations
in both the percentage of phagocytes associating with AMA and the number
of organisms per 100 host cells (Table I). In the two repeat titrations
using MPM, these effects were first detectable with either 0.1 or 1 pg
LF/ml. However, because the concentration of LF producing a maximal
enhancement was 10 ug/ml, it was used in subsequent experiments. At 50
pg LF/ml, there was no significant change in the extent of AMA-MPM
association with respect to the control values.

To establish whether LF enhanced AMA-MPM interaction through an
effect on the host cell or on the AMA, or on both, experiments were
designed in which each of these cells was treated with LF, washed and then
co-cultured with the AMA with 10 pg LF/ml for various periods of time
significantly increased their association (Table II). The minimal MPM
pretreatment time required to significantly increase both the percentage

of AMA-associated MPM and the number of AMA per 100 MPM was found to be 30

44

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45
Footnotes to Table I

a This set of results is typically representative of two separate
experiments with the same protocol. The experiments with MPM and HBM were
carried out separately.

b %C, percentage of change with respect to the corresponding
control value (no LF present).

c The difference between this value and its corresponding control

value is statistically significant (P<0.05).

TABLE II

Effects of pretreatment of MPM or AMA with LE on their capacity to

associate with the untreated counterpart°

 

Pretreated Pretreatment Pretreatment %MPM containing

Hue:- ofAMA

 

cell tine (m) AMA m1" per 100 m (it)
new 012141051 10 10.0 1 1.0 20.0 1 0.0

ion 10‘ 10 10.2 1 0.0 ( 1.31 24.0 11.5 (20.0)
14914 0190054 20 15.2 1 0.0 19.0 1 0.7

m 12 20 10.7 1 0.0 1 9.9) 29.5 1 0.5" 155.3)
mm 00501001 30 10.2 1 0.5 20.5 1 0.0

mi 12 30 20.0 1 0.7‘ (20.4) 37.3 1 0.0" (01.01
mm 01451054 00 15.2 1 0.0 20.7 1 0.5

new LF 00 22.0 1 0.0‘ (50.0) 30.3 1 0.7“ (05.01
7044 01010054 10 10.0 1 1.0 22.3 1 0.9

4144 u: 10 23.5 1 2.5 (25.01 33.0 1 3.3‘1 (50.71
m 074210051 20 10.0 1 2.3 22.7 1 2.7

m LE 20 31.5 11.0‘ (07.51 45.5 1 2.2‘I (100.4)
m ocmsn 30 20.0 1 1.3 25.5 1 3.4

m 10 30 30.0 1 2.3d (04.01 55.0 1 2.7" (110.01
7044 012141050 00 10.5 1 1.0 25.0 1 1.9

m 10 00 30.0 1 0.0" (123.01 00.0 1 2.0" (135.71

 

For footnotes please see next page.

... . .—..-.-.-——-

47

Footnotes to Table II

a This set of results is typically representative of three

separate experiments with the same protocol.

b %C, percentage of change with respect to the corresponding

control value (DMEM+BSA).

c In these experiments LF was always used at a final concentration

of 10 pg/ml. The experiments in which MPM and AMA were pretreated with
LF were carried out separately.
d The difference between this value and its corresponding control

value is statistically significant (P<0.05).

48

min whereas for the AMA it was 10 min (i.e., when first tested).

Next we set out to establish whether HBM, MPM or AMA bound LF. The
results of immunofluorescence tests using fluorescein-labeled anti-LP 190
showed that, although untreated HBM, MPM and AMA did not have detectable
LF on their surface, approximately 60% of the HBM, 80% of the MPM and 50%
of the AMA bound the glycoprotein when the latter was present at 100
Pgflml (Table III). Shnilar tests performed with 10 pg/LF/ml, i.e., the
concentration of LF used in our parasite-host cell interaction assays,
detected lower but significant percentages of LF-positive HBM, MPM and
similar percentages of LF-positive AMA. The presence of receptors for LF
on the surface of macrophages and monocytes has been previously
demonstrated (18, 19). The results of experiments in which we measured

125

concentration-dependent binding of I-LF to AMA revealed that LF

125

binding reached saturation levels when 96 ng I-LF was bound to 1.2 X

106 AMA (Fig. 1). Through Scatchard analysis, the data was found to

correspond to 1.06 X 106 LF receptors per AMA and Ka= 3.0 X 105

l.mol'1. To test the specificity of the 125

I-LF binding to the
parasites we carried out competition type of experiments adding unlabeled
LF; control experiments were included in which unlabeled hemoglobin was
added instead of LF. In the presence of a 13-molar excess of cold LF, a

79% reduction in the uptake of 125

I-LF uptake was observed (from 29,199
1_2,766 to 6,100 :_1,732 cpm) whereas the reduction caused by
hemoglobin was insignificant (from 29,199 :_2,766 to 27,721 1 1,879
cpm).

Inhibition of the LF.enhancigg effect by agents which bind LF. To

establish whether binding of LF to MPM or AMA was required to produce the

49

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50
Footnotes to Table III
a This set of results is typically representative of two separate
experiments with the same protocol. All of the differences between the

experimental values and the corresponding control values are statistically

significant (P<0.05).

SI

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53

enhancing effect, we incorporated into the system an agent known to bind
LF. Selected for this purpose were anti-LF IgG and oc-MM (18). Five
different protocols were designed to clarify not only if LF binding to the
interacting cells was a requirement but also if blocking LF bound to MPM
or AMA would prevent the enhancing effect (Table IV). As can be seen in
Table V, co-cultures of MPM and AMA incubated with LF together with either
anti-LF IgG or oc-MM (protocol No. 1, see Materials and Methods) resulted
in a marked reduction in the extent of the LF enhancing effect. Neither
anti-LE nor oc-MM affected AMA-macrophage interaction in the absence of
LF. Although Table V, shows results obtained with 0.25M oc-MM, significant
inhibitory effects were also produced by concentrations of this reagent as
low as 0.01M (data not shown). Hhen MPM cultures were pretreated with LF
and then incubated with the blocking agents before receiving untreated AMA
(protocol No. 2), no inhibition of the LF effect was seen. In contrast,
when MPM cultures were simultaneously incubated with LF and anti-LF (or 0:
-MM), washed and then co-cultured with untreated AMA (protocol No. 3), the
enhancing effect of LF was not seen. The LF effect was markedly inhibited
when AMA were pretreated first with LF, then with anti-LF or oc-MM and
finally incubated with untreated MPM cultures (Table V, protocol No. 4).
The LP effect was also abrogated when AMA were incubated with LP in the
presence of either anti-LF or oc-MM prior to being added to the MPM
cultures (protocol No. 5). Hhen normal rabbit IgG was substituted for
rabbit anti-LF 196, the LF effect was not altered significantly in any of
the five protocols and normal rabbit IgG had no detectable effect of its
own on MPM-AMA association. It is noteworthy that, in all five protocols

and in all repeat experiments, the changes in the percentage of MPM

54

TABLE IV

Summary of protocols 1 through 5

 

Protocol No. 1

a) [MPM + AMA + LF + anti-LF IgG (or o:-MM)J (2 hr at 37°C) >

 

b) terminate by washing and fixation
Protocol No. 2

a) [MPM + LF] (30 min, 37°C) >

b) wash, add anti-LF IgG (or oz-MM) (30 min, 37°C) >
(2 hr at 37°C) >

 

c) wash, add untreated AMA

 

d) terminate by washing and fixation

Protocol No.3

a) [MPM + LF + anti-LF IgG (or oc-MM)] ‘30 "‘1": 37°“ >

 

b) wash, add untreated AMA (2 hr at 37°C) >

 

c) terminate by washing and fixation

Protocol No.4

a) [AMA + LF] (30 Ni", 37°C) >

 

(30 min, 37°C) >
(2 hr at 37°C)

b) wash, add anti-LF IgG (or oc-MM)

 

c) wash, add these AMA to untreated MPM cultures
d) terminate by washing and fixation
Protocol No.5

a) [AMA + LF + anti-LF 190 (or oc-MM)] ‘30 “1": 37°C’ >

 

b) wash, add these AMA to untreated MPM cultures (2 hr at 37°C)

c) terminate by washing and fixation

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56

Footnotes to Table V

a The sets of results for each protocol are typically
representative of two to three separate experiments with the same
protocol. For protocol No. 1, the experiments with oc-MM and anti-LF were
carried out separately. For the description of protocols No. 1, 2 and 3
see Materials and Methods.

b The final concentrations of LF, oc-MM, anti-LF IgG and normal
rabbit 196 used in these experiments were 10 pg/ml, 0.25M, 3.3 mg/ml and
3.3 mg/ml, respectively.

c See footnote b_under Table II.

d The difference between this value and the corresponding control

(DMEM+BSA) was statistically significant (P<0.05).

57

containing AMA were of the same direction as those shown in Table V and VI
for the number of AMA per 100 MPM (data not shown).

Effects of LE on latex bead uptake by MPM. To determine if the
enhancing effect of LF on MPM was unique for AMA, we performed identical
experiments using latex beads. The results indicated that the presence of
LF during incubation of MPM with the beads or LF treatment of the MPM
prior to adding the particles significantly enhanced particle uptake
(Table VII).

Effects of LF on AMA killing by HBM or MPM. Hhile the results

 

presented above demonstrate the increased capacity of LF-treated MPM to
take up AMA and latex particles, they did not reveal whether LF-treated
MPM would also di5play increased killing capacity. To test this
possibility, we set up experiments with HBM or MPM in which 10 pg LF/ml
was present during the 2-hr cell-parasite interaction period and monitored
the decrease in parasite load 4 hr after removing both LF and the
non-bound AMA. Selection of the 4-hr incubation period was based on
previous results revealing that untreated MPM destroyed a small number of
AMA 6 hr after initiation of the MPM-AMA interaction (2). The results
presented in Table VIII showed that HBM and MPM destroyed significantly
larger numbers of AMA after exposed to LF. In separate experiments, MPM
that had been pretreated with LF for 60 min and then incubated with'
untreated AMA for 2 hr cleared a larger number of parasites than
mock-treated MPM over the 4-hr period (Table IX). At this point, we were
curious to find out whether pretreatment of the AMA with LF would also
lead to their more rapid destruction by untreated MPM. The results

presented in Table VIII showed that this was indeed the case. Of

58

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59

Footnotes to Table VI

a The sets of results for each protocol are typically
representative of two to three separate experiments with the same
protocol. For the results obtained when LF was present during AMAAMPM
interaction, see Table IV, protocol No. 1. For the description of
protocols 4 and 5 see Materials and Methods.

b,c,d See footnotes b,c,d under Table IV-

60

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. 61

Footnotes to Table VII

a Each set of results is typically representative of two separate
experiments with the same protbcol.

b Step A consisted of incubating MPM cultures with the indicated
solution at 37°C for 30 min and was immediately followed by step 8,
which consisted of incubating these MPM with the indicated solution plus
latex beads for 2 hr.

c See footnote b_under Table 11.

d

The difference between this value and the corresponding control

(DMEM+BSA) is statistically significant (P<0.05).

62

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63

Footnotes to Table VIII

a This set of results is typically representative of two separate
experiments with the same protocol.

b DMEM+$A alone or containing 10 ug LF/ml. LF was present during
the 2-hr co-culture of phagocytic cells with AMA.

c The percentage of AMA killed during the 4-hr period was

calculated by the equation:
1 AMA killed 3 2-hr value - 6-hr value X 100.

2-hr value

d Values given under the 2- and 6-hr columns were measured in

cultures terminated immediately after removal of the free AMA (i.e.,
after 2 hr of AMA-MPM interaction) and 4 hr later, respectively.

e See footnote 9 under Table II.

f The difference between this value and the corresponding control
was statistically significant (P<0.05).

9 This extent of killing was_statistically different from that seen

with cells mock-treated with DMEM+BSA (P<0.05).

64

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65

particular interest was the observation that untreated MPM that had taken
up untreated AMA also killed the organisms at a faster rate if LF was
added to the culture (Table X). It is noteworthy that, in all of the
experiments designed to monitor AMA killing, free AMA were virtually
undetectable in the culture medium at the end of the experiment, rendering
unlikely the possibility that reductions in parasite load could have
resulted from detachment of surface-bound organisms.

Mechanism of killing of AMA by LF—treated MPM. A recent report

 

from our laboratory has documented that the killing of T. cruzi AMA by
MPM involves H202 (2). To test whether LF-treated MPM killed the
parasites by a similar mechanism, we set up experiments using scavengers
of oxygen reduction intermediates. Catalase (scavenger of M202) did

not affect the enhancing effect of LF on MPM-AMA association but
completely abrogated parasite destruction (Table XI). Heat-inactivated
(100°C, 20 min) catalase did not inhibit killing. Histidine (scavenger
of 102) and $00 (scavenger of 02’) had no effect on AMA uptake by
LF-treated MPM but partially inhibited killing. Heat-inactivated SOD had
no effect on either parameter. That 027 was indeed produced by MPM

that had internalized AMA was confirmed by a) the observation that, among
the AMA-containing MPM, 83% of the cells gave a positive nitroblue
tetrazolium reduction test whereas only 44% were positive if mock-treated
with medium and b) the finding that only 15% and 12% of these cells,
respectively, gave a positive nitroblue tetrazolium test if 500 was

present before and during cell-parasite interaction.

66

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8cm___8 <z< u <z< co amass: co =8a=m8 cw8ca tacos =o_8=_om

 

 

achVVV8 muVmacaN

 

co <z< N=V=V~8=ou 2a: co mwc=8V=u o8 a8 co coV8Vuua cc Vuaccw
x N8N<V

67

Footnotes to Table x

a This set of results is typically representative of two separate
experiments with the same protocol.

b Percentage of reduction in parasite load during the 4-hr period
(reference value 27.8).

‘ LF (10 pg/ml) or 05mm was added immediately after removal of
the free AMA and left in the cultures for the indicated amounts of time.
After removing the LF by washing, the cultures were incubated with fresh
DMEM+FBS for the remainder of the 4-hr period.

d The value shown in this line was obtained immediately after
removal of the free AMA, i.e., after 2 hr of co-culture of MPM and AMA.
All other values in this table were obtained 4 hr later.

e This extent of killing was statistically different from that
produced by cells mock-treated with DMEM+$A (P<0.05).

 

 

 

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69

Footnotes to Table XI:
a This set of results is typically representative of two separate
experiments with the same protocol.

b This percentage represents the reduction in parasite load during

the 4-hr period.

c The difference between this value and the corresponding control
(DMEM+BSA) is statistically significant (P<0.05). In each experiment, all
LF effects (Z-hr values) are statistically comparable.

d The difference between this extent of killing and that seen in
the control (DMEM+BSA) is statistically significant (P<0.05).

e The difference between this extent of killing and that seen with

LF alone is not statistically significant (P<0.05).

70

DISCUSSION

These results show that LF increases the uptake and intracellular
destruction of intracellular forms of a protozoan pathogen by HBM or MPM.
Concentrations of LF enhancing the interaction of the phagocytic cells
with T. cruzi AMA ranged from 0.1 to 10 ug/ml. These levels compare
with normal human plasma concentrations of 1.5 11.8 ug/ml (20), and
pathological levels varying between 4 and 28 ug/ml in cases of burn
injury (21), 12 to 22 ug/ml in cases of chronic myeloid leukemia in
relapse and 5 and 12 ugflml in two cases of hypersplenism secondary to
hepatic cirrhosis (20). At higher concentrations -50 pg/ml- LF did not
produce an enhancing effect (Table I) and we thought this might owe to
saturation of LF receptors on the surface of MPM and AMA and that at
lower, effective concentrations, LF might bridge the interacting cells.
For this hypothesis to be valid two conditions had to be met: i) LF would
have to bind to the surface of both the AMA and the phagocytes and ii)
pretreatment of either MPM or AMA would have to increase association of
the treated cell with the untreated counterpart. The results of our
immunofluorescence studies showed that HBM, MPM and AMA indeed could bind
LF. Of note, the results obtained with HBM and MPM are in agreement with
those of other investigators who reported that radiolabeled LF binds to
HBM as well as to MPM (15, 19, 22-23). The results of the experiments in

125

which we measured concentration-dependent binding of I-LF to AMA and

determined the ability of cold LP to compete with radiolabeled LF for

binding sites on the parasites indicated that LF bound specific receptors

on the AMA surface. As to the second condition, the pretreatment of

71

either AMA or MPM with LF did result in significantly enhanced association
of these cells with the untreated counterpart. Hhile these results
appeared to support the hypothesis that LF bridged phagocytes and AMA,
additional experiments were carried out to confirm it. If LF increased
the association merely by bridging MPM and AMA, blocking agents such as
anti-LP and oc-MM would be expected to inhibit at least in part the effect
both when present during LF incubation with either cell and when used to
treat MPM or AMA that had already been treated with LF (i.e., immediately
before being exposed to the untreated counterpart). The results showed
that, whenever the experimental conditions allowed the blocking of LP by
oeMM or anti-LF IgG in the fluid phase (i.e., during cell treatment;
protocols No. 1, 3 and 5), AMA-MPM association was indeed inhibited
(Tables V and VI). However, when MPM were pretreated with LF, washed, and
then incubated with either blocking agent (protocol No. 2), the LF effect
was not significantly inhibited. This observation implied that LF binding
to the MPM surface was necessary for this cell to display the enhancing
effect and that subsequent treatment with either blocking agent could not
inhibit it or reverse it. Because bridging in protocol No. 2 should have
been substantially prevented by anti-LF or oc-MM but was not, we surmised
that bridging was probably not the major factor underlying the LF effect.
In comparing the results obtained with all five protocols we noticed
that the LF effect had been seen every time that the MPM had been directly
exposed to LF either present in the fluid phase (even if blocked
afterwards) or unhindered on the AMA surface. The notion that LF contact
with the MPM was required to produce the enhancing effect was supported by

the results of protocol No. 4, showing that blocking LF bound to AMA prior

72

to addition to the MPM cultures abrogated the LF effect (Table VI).
Hence, we formulated a new hypothesis, that LF activated MPM, and
proceeded to test it. First, we observed that MPM treated with LF either
prior to or during incubation with latex beads took up significantly
greater numbers of particles than did mock-treated MPM (Table VII). There
was also an increase in the proportion of MPM taking up latex beads.

An independent indication of HBM or MPM activation by LF was
provided by the results of the experiments in which parasite killing was
monitored, particularly under a condition (6-hr cell-parasite interaction
period) allowing minimal AMA killing by untreated HBM or MPM (2). Both
HBM and MPM disposed of relatively large numbers of AMA, significantly
greater than those killed by mock-treated cells, regardless of whether LF
was present during the initial Z-hr cell-parasite interaction (Table VIII)
or used only to treat MPM or AMA prior to their interaction with the
untreated counterpart (Table IX). The large, significant difference
between the extents of AMA killed by HBM or MPM exposed to LP (>75!) and
by mock-treated phagocytes ((16.4%) denoted the ability of LF to increase
the cytolytic capacities of HBM and MPM. A similar increase in cytolysis
was seen when untreated MPM were exposed to AMA which had been treated
with LF in an otherwise identical experiment (Table IX). Consequently,
the MPM appeared to mount the LF effects (i.e., for uptake and killing)
regardless of whether the glycoprotein was presented to them in the fluid
phase or bound to the AMA surface.

If indeed LF activates MPM, untreated MPM which have already
engulfed AMA would be expected to display an increased killing capacity

after treated with the glycoprotein. This was in fact the case as

73

evidenced by the greater percentages of parasites killed by AMA-containing
MPM when they were treated with LF (Table X). These results, together
with those of Ambruso and Johnston (24), who postulated that LF can
activate neutrophils, make it tempting to speculate that LF may be a
common activator of different types of inflammatory cells.

Although activation of MPM by LF might by itself account for the
entire LF effect, a possible contributory role of LF bridging can not be
totally ruled out and the lack of effect of 50 ug LF/ml (Table 1) remains
to be explained. Conceivably, this high concentration could be
supraoptimal, reminiscent of the lack of effect of excess doses of
concanavalin A on lymphocyte proliferative responses.

The results of experiments in which scavengers of intermediates of
oxygen reduction were present in cultures before, during and after MPM
interaction with AMA indicated that H202 was the chemical species

mediating most of the parasite killing, although 02‘ and 1

02 also
played a role (Table XI). Previous studies with unelicited MPM showed
that these cells could kill T. cruzi AMA via H202 and that neither
SOD (scavenger of 02‘) nor histidine (scavenger of 102) had

significant inhibitory effects (2). The present results, showing that the
latter two oxygen metabolites were also involved in AMA killing by
LF-treated MPM, suggest that additional cytolytic resources available to
MPM could be recruited to the action upon LF stimulation. Production of
02‘ by LF-treated MPM that had internalized AMA was also evidenced by

the reduction of nitroblue tetrazolium to formazan; the percentage of

cells containing AMA and formazan among LF-treated MPM was much greater

than among mock treated MPM. Interestingly, LF has been shown to induce

74

production of OH’in human neutrophils (24).

LF shares with fibronectin (8), another glycoprotein produced by an
inflammatory cell, an ability to enhance macrophage interaction with I;_
25251. Conceivably, other proteins of inflammatory cell origin may play
a role in the uptake and clearance of T. cruzi and perhaps other
microorganisms as well, contributing to host resistance mechanisms against

infections.

75

REFERENCES

Carvalho, R. M., M. N. L.Meirelles, H. de Sousa, and N. Leon.

1981. Isolation of the intracellular stage of Trypanosoma cruzi

 

and its interaction with mouse macrophages in. vitro. Infect.

Immun.33:546.

 

Villalta, F., and F. Kierszenbaum. 1984. Role of inflammatory cells
in Chagas' disease. II. Interactions of mouse macrophages and human
monocytes with intracellular forms of Trypanosoma cruzi: uptake

and mechanism of destruction. J. Immunol. 133:3338.

Kierszenbaum, F., E. Knecht, 0. B. Budzko, and M. C. Pizzimenti.
1974. Phagocytosis: a defense mechanism against infection with

Trypanosoma cruzi. J. Immunol. 112:1839.

 

 

Williams, 0. M., and J. 5. Remington. 1977. Effects of human
monocytes and macrophages on Trypanosoma cruzi.Immunology

Villalta, F., and F. Kierszenbaum. 1983. Role of polymorphonuclear
cells in Chagas' disease. I. Uptake and mechanisms of destruction

of intracellular (amastigote) forms of Trypanosoma cruzi by human

 

neutrophils. J. Immunol. 131:1504.

 

Villalta, F., and F. Kierszenbaum. 1984. Role of inflammatory cells
in Chagas' disease. I. Uptake and mechanisms of destruction of
intracellular (amastigote) forms of Trypanosoma cruzi by human

eosinophils. J. Immunol. 132:2953.

 

Mosesson, M. H., and D. L. Amrani. 1980. The structure and biologic

activities of plasma fibronectins. Blood 56:145.

 

10.

11.

12.

13.

14.

15.

76

Hirth, J. J., and F. Kierszenbaum. 1984. Fibronectin enhances
macrophage association with invasive forms of Trypanosoma cruzi.

J. Immunol. 133:460.

 

 

Romana, C. 1963. Enfermedad de Chagas. Lopez Libreros Editores,
Buenos Aires, P. 97.

Spitznagel, J. K. 1984. Nonoxidative antimicrobial reactions of
leukocytes. In Regulation of Leukocyte Function. Edited by R.
Snyderman. Plenum Publishing Corp., New York. P. 283.

Laffrell, M. S., and J. K. Spitznagel. 1974. Intracellular and
extracellular degranulation of human polymorphonuclear azurophil and
specific granules induced by hmnune complexes. Infect.Immun.

10:1241.

 

Pryzwansky, K. B., E. K. Macrae, J. K. Spitznagel, and M. H. Cooney.
1979. Early degranulation of human neutrophils: immunocytochemical
studies of surface and intracellular phagocytic events. §g11_
18:1025.

Hansen, N. E., H. Karle, V. Andersen, J. Malmquist, and G. E. Hoff.
1976. Neutrophilic granulocytes in acute bacterial infection.
Sequential studies on lysozyme, myeloperoxidase and lactoferrin.

Clin. Exp.Immunol. 26:463.

 

Nishiya, K., and D. A. Horwitz. 1982. Contrasting effects of
lactoferrin on human lymphocyte and monocyte natural killer activity
and antibody-dependent cell-mediated cytotoxicity. J. Immunol.
129:2519.

 

Villalta, F., and F. Kierszenbaum. 1982. Growth of isolated

amastigotes of Trypanosoma cruzi in cell-free medium.

J.Frotozool. 29:570.

16.

17.

18.

19.

20.

21.

22.

23.

77

Villalta, F., and F. Kierszenbaum. 1983. Role of cell surface

mannose residues in host cell invasion by Trypanosoma cruzi.

 

Biochim. Biophys. Acta 736:39.

Murray, H. H., and Z. A. Cohn. 1980. Macr0phage oxygen-dependent
antimicrobial activity. III. Enhanced oxidative metabolism as an
expression of macrophage activation. J. Exp. Med. 152:1596.
Bennett, R. M., and J. Davis. 1981. Lactoferrin binding to human
peripheral blood cells: an interaction with a B-enriched population
of lymphocytes and a subpopulation of adherent mononuclear cells.
J.Immunol. 127:1211.

Van Snick, J. R., and P. L. Masson. 1976. The binding of human
lactoferrin to mouse peritoneal cells. J. Exp. Med. 144:1568.
Bennett, R. M., and C. Mohla. 1976. A solid phase radioimmunoassay
for the measurement of lactoferrin in human plasma and disease.

J. Lab. Clin. Med. 88:156.

Holach, B., T. 0. Coates, T. E. Hugli, R. L. Baehner and L. A. Boxer.
1984. Plasma lactoferrin reflects granulocyte activation in burn
patients. J. Lab. Clin. Med. 103:264.

Van Snick, J. L., P. L. Masson, and J. F. Heremans. 1974. The
involvement of lactoferrin in the hyposideremia of acute

inflammation. J. Exp. Med. 140:168.

 

Imber, M. J., S. V. Pizzo, H. J. Johnson, and D. 0. Adams. 1982.
Selective diminution of the binding of mannose by murine macrophages

in the late stages of activation. J. Biol. Chem. 257:5129.

 

78

24. Ambruso, D. R., and R. B. Jonhston. 1981. Lactoferrin enhances
hydroxyl radical production by human neutrophils, neutrophil

particulate fractions, and an enzymatic generating system. J. Clin
Invest. 67:352.

79

GMWERII

LACTOFERRIN EFFECTS ON THE INTERACTION OF BLOOD FORMS OF
Trypanosoma cruzi HITH MONONUCLEAR PHAGOCYTES

 

ABSTRACT

Pretreatment of mouse peritoneal macrophages (MPM) or human blood
monocytes (HBM) with lactoferrin (LF) -a glycoprotein secreted by
neutrophils, whose levels increase in inflammatory conditions- modified
the outcome of the interaction between these cells and blood
(trypomastigote) forms of Trypanosoma cruzi. A 24-hr LF pretreatment
was required for MPM to display an increased capacity to take up the
organisms whereas HBM -whose density of surface LF receptors is greater
than that of MPM- required a pretreatment of only 2 hr. Since the extent
of trypomastigote incorporation by untreated MPM was not significantly
affected by treating the former with LF, and indirect immunofluorescence
tests failed to reveal the presence of LF receptors on the surface of
these organisms, it appeared that the effect of LF was mostly on MPM.

Hhen MPM were treated with LF for 2 hr and then exposed to the-
trypomastigotes for 2 hr, the numbers of parasites per 100 MPH recorded 70
hr later were smaller than those of mock-treated MPM, but there was no
significant difference in the proportions of MPM taking up parasites. MPM
which had ingested trypomastigotes and were then treated with LF also
cleared the organisms faster than mock-treated MPM. Lactoferrin
pretreatment also increased the trypanocidal capacity of HBM. H202

was involved in parasite killing by LF-treated MPM since catalase
prevented it. These results show that a neutrophil product can facilitate
the uptake and disposal of blood forms of T. cruzi by'mononuclear
phagocytes and suggest that LF may influence host defense mechanisms

against infection with this parasite.

81

INTRODUCTION

Although inflammatory cell infiltration is one of the early
developments in the pathology of Chagas' disease (reviewed by Andrade 6
Andrade, 1979 and by Romaha, 1963), its precise role in host defense or
pathogenesis remains to be defined. Several investigators have examined
interactions between some types of inflammatory cells and mammalian forms
of Trypanosoma cruzi. This yielded information about the ability of
macrophages or monocytes to take up and destroy bloodstream (trypomastigo—
te)(Alcantara 6 Brener, 1980; De Almeida Maria, Alcantara 6 Brener, 1982;
Kierszenbaum, Knecht, Budzko 6 Pizzimenti, 1974; Nathan, Nogueira,
Juangbhanich, Ellis 6 Cohn, 1979; reviewed by Scott 6 Snary, 1982) and
intracellular (amastigote) forms of the parasite (Carvalho, Meirelles, De
Souza 6 Leon, 1981; Villalta 6 Kierszenbaum, 1984, 1984a), and the
capacity of eosinophils and neutrophils to destroy these organisms via
antibody-dependent cell-mediated cytotoxicity (Kierszenbaum, 1979;
Kierszenbaum 6 Hayes, 1980; Okabe, Kipnis, Calich 6 Dias da Silva, 1980)
or after phagocytosis (Kierszenbaum gt_gl., 1974; Kierszenbaum, Villalta
6 Tai, 1986). There have also been reports concerning biochemical factors
and reactions involved in the uptake of blood forms of T. cruzi by
macrophages (Connelly 6 Kierszenbaum, 1984, 1985; Villalta 6 Kierszenbaum,
1983, 1983a; Hirth 6 Kierszenbaum, 1982, 1983). Some of our recent
efforts have focused on proteins associated with inflammatory conditions
and their ability to modulate the outcome of the interaction of the two
mammalian forms of T. cruzi with inflammatory-type cells (Hirth 6

Kierszenbaum, 1984; Villalta 6 Kierszenbaum, 1986). He first examined the

82

effects of lactoferrin (LF) -an iron-binding glycoprotein produced by
neutrophils in increased amounts during inflammatory conditions
(Pryswansky, Macrae, Spitznagel 6 Cooney, 1979)- on the fate of
amastigotes in mononuclear phagocytic cells (Lflna 6 Kierszenbaum, 1985).
In this paper we report the effects of LF on mononuclear phagocyte

interaction with the trypomastigote form.

83

MATERIALS AND METHODS

Animals. Four to six-week-old Crl:CD-1(ICR)BR Swiss mice were purchased
from Charles River Laboratory (Portage, Michigan).

T. cruzi. Tulahuéh strain trypomastigotes were isolated from blood of
mice infected intraperitoneally 2 weeks previously with 2 x 105
trypomastigotes. The organisms were separated from the blood cells by
density gradient centrifugation over Isolymph (Ballard Schlesinger, Carle
Place, New York) (Budzko 6 Kierszenbaum, 1974) followed by chromatography
through a diethylaminoethyl-cellulose column (Villalta 6 Leon, 1979). The
flagellates were washed with Dulbecco's modified minimal essential medium
containing penicillin (100 units/ml) and streptomycin (100 pg/ml) (DMEM)
and resuspended at 1 X 107 organisms/ml in the same medium supplemented
with either 1% bovine serum albumin (Sigma Chemical C0., St. Louis,
Missouri) (DMEM+BSA) or 10% fetal bovine serum (Gibco, Grand Island, New
York) (DMEM+FBS). Parasite viability was always >99.8%.

Mouse peritoneal macrophages (MPM). Unstimulated mice were sacrificed

by excess ether inhalation. The methods for collecting the resident
peritoneal macrophages and for setting up monolayers of these cells on
Lab-Tek microscope slide tissue culture chambers have been described in
detail elsewhere (Zenian 6 Kierszenbaum, 1982). These monolayers were
incubated at 37°C for 18 hr in a 5% COZ-in-air incubator (saturated

with water vapor) and washed immediately before being subjected to the
appropriate treatment. These cultures consisted of >98%

nonspecific-esterase-positive cells with typical macrophage morphology.

Human blood monocytes (HBM). Blood was drawn from healthy donors. The

methods to purify monocytes and to prepare adherent monolayers on Lab-Tek.
tissue culture chambers have been described (Villalta 6 Kierszenbaum,
1984a). These monolayers were incubated at 37°C for 2 hr in a 5% C02
incubator and washed with DMEM prior to further treatments. These cultures
consisted of>99%.nonspecific-esterase-positive, trypan-blue-excluding
monocytes.

Pretreatment of MPM or HBM with LF. Monolayers of MPM or HBM were

 

incubated at 37°C for variable periods of time with 0.3 ml of DMEM+FBS
alone or containing varying concentrations of LF (see Results). Unless
otherwise stated, LF was removed from the cultures by three washings with
DMEM prior to incorporating T. cruzi into the cultures (see below). The
batches of human LF (at 10% iron saturation) were purchased from Sigma.

Pretreatment of trypomastigotes with LF. Nine volumes of parasite

 

suspension were mixed with one volume of DMEM+BSA alone or containing LF
solution at ten times the desired final concentration in the reaction
mixtures and incubated at 37°C for 2 hr . After washing the trypanosomes
twice with DMEM by centrifugation (800 x G, 4°C, 20 min), they were
resuspended in DMEM+BSA at l X 107organisms lml.

Determination of T. cruzi uptake by MPM or HBM. All experimental and

 

 

control conditions were tested in triplicate. Lactoferrin-treated or
mock-treated MPM or HBM monolayers received 0.3 ml of the appropriate
parasite suspension and incubated at 37°C and 5% CO2 for 2 hr . After
removing the free parasites by three washings with DMEM, the cultures were
fixed with absolute methanol and stained with Giemsa. Not less than 200

cells were screened microscopically (X1000), recording the total number of

screened cells, the number of parasites associated with the screened cells

85

and the number of MPM or HBM associated with one or more trypomastigotes.
These values were used to calculate the percentage of MPM or HBM with
parasites and the average number of organisms per 100 cells.
Determination of parasite killing by MPM or HBM. Co-cultures of :1;
55251 and MPM or HBM were incubated at 37°C and 5% CO2 for 2 hr as
described in the preceding subsection. After removing the parasites, 0.3
ml of fresh DMEM+FBS was added and the cell monolayers were further
incubated under the same'conditions for various periods of time. These
cultures were terminated and processed for counting as described above.
All tests were performed in triplicate. In some experiments, untreated
MPM which had already internalized untreated parasites were tested for
their capacity to kill the latter after LF was added to the culture
medium. In this case, MPM monolayers were co-cultured with the parasites
as described above and, after removing the free flagellates, 0.3 ml of
DMEM+FBS alone or containing 10 pg LF/ml was added. Replicates of these
cultures were then incubated at 37°C for various periods of time, washed
with DIEM and terminated by fixation as described above. In some
experiments, catalase (recrystallized beef liver catalase, Sigma) was
added in 0.4 ml MM+FBS to attain a final concentration in the cultures
of 160,000 units/ml and remained present in the culture medium throughout
the incubation period. Heat-inactivated (100°C, 20 min) catalase was
used in control assays.

Immunofluorescence assay for LF. Binding of LP by T. cruzi
trypomastigotes was tested by incubating parasites fixed with 0.25%
formaldehyde in phOSphate-buffered saline pH 7.0 (PBS) smeared on

microscope slides with solutions containing 10 or 100 ug LF/ml in

86

DMEM+BSA for 2 hr . After washing three times with PBS, the smears were
incubated with fluorescein-labeled rabbit anti-human LF 196 (Cappel
Laboratories, West Chester, Pennsylvania) at 37°C for 30 min, washed

again with PBS, air dried, and examined by fluorescence microscopy.
Presentation of results and statistics. All results are expressed as

the mean :_1 5.0. Differences were considered to be significant if

P50.05 as determined by Student's “t“ test. The sets of results

presented in the tables are typically representative of two to four repeat

experiments.

87

RESULTS

Effects of LF on the uptake of T. cruzi trypomastigotes by MPM or
flgfl;Pretreatent of MPM with 10 pg LF/ml altered their capacity to take

up blood forms of T. cruzi as revealed by increases in both the

percentage of parasite-containing MPM and the average number of organisms
per 100 MPM; these increases were seen immediately after removing the
non-bound parasites (i.e., at 0 hr) (Table 1). Although this effect was
consistently produced by a 24-hr pretreatment of the MPM with LF, a
relatively small but nevertheless significant increase was seen in some
experiments (e.g., the one represented in Table 1) after a 12-hr
pretreatment. Experiments performed with HBM showed that these cells were
also capable of greater parasite uptake when pretreated with LF but, in
this case, the effect was seen after pretreating the HBM with either 10 or
100 pg LF/ml for only 2 hr (Table 2, results obtained at 0 hr).

No significant change in the extent of parasite interaction with
untreated MPM was seen when the trypomastigotes were pretreated with up to
100 pg LF/ml for 2 hr (data not shown). This negative result led us to
test whether or not the parasite would bind LF. The results of indirect
immunofluorescence tests failed to produce any evidence of such binding
when the flagellates were incubated with up to 100 pg LF/ml.

Effects of LF on trypomastigote killing by MPM or HBM. The results of
experiments in which the MPM were pretreated with 10 pg LF/ml for 2 hr,
washed, and then exposed to T. cruzi for 2 hr in the absence of LF,
showed that the number of parasites per 100 MPM was significantly smaller

than that of mock treated MPM when measured 70 hr later, but not 22 or

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89

Footnotes to table 1

MPM were incubated with LF for 6, 12 or 24 h, washed and
cultured with the parasites for 2 h. After removing the
non-bound organisms, one third of the MPM cultures were fixed
whereas the remaining cultures received fresh medium and were
incubated for an additional 10 or 22 h.

(%C), percent increase in parasite uptake by LF-treated MPM
(calculated with respect to the value obtained in the absence of
LF). A %C value is shown where it was statistically significant
(P50.05).

{ZR}, percent reduction in parasite load calculated with reSpect
“to the corresponding value obtained immediately after removing
the non-bound organisms (O-h values); a SR value is shown where

it was statistically significant (P50.05).

90

.H apnop Luca: muuocuoo» mom 0..
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umuonauc. mew: was E=.uoe gmm.» vo>.mum. upo; nguo mga moueogz A; o. xpuuo.umes.
aux.» 6cm: mmcaupau 2m: ms» mo mpg; .msm.camco acaoauco: mg» u:.>oswc gwum< .z N

so» www.mncoa mg» sum: copay—nu use uwgmox .: H use co» m. gu.) vaunnaucw mew: so:

 

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..N u 9.... .5 .+. o... a... H .2... . 3 u «.2 Sims:

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.2 so

 

 

23.8 .50 mice... 83¢ .35. oz< a: 32k
ow >h~u<a<u «aux» zo m4 :hnz in: go hzm2h<mmbmma mo mpgmmmu u N m4m<p

91

46 hr later (Table 3).However, the percentages of infected MPH showed
insignificant variations during the 70-hr period. Similar results were
obtained when 10 pg LF/ml was present in the MPM cultures during the 2-hr
period that these cells were exposed to the trypomastigotes (Fig. 1).
Because in our experimental system free parasites usually appear in the
culture medium approximately 4 days after MPM infection (Lima and
Kierszenbaum, unpublished results), and this would have complicated the
interpretation of the results, measurements were not made after 70 hr.

In addition to enhancing parasite uptake by MPM or HBM, pretreatment
of MPM with 10 pg LF/ml for 24 hr and of HBM with 10 or 100 pg LF/ml for
2 hr increased their cytotoxic capacities (Table 1, results obtained
after 10 or 22 hr and Table 2, results obtained after 10 hr). As seen in
Table 1, the reductions in the parasite contents of LF-treated MPM over
the 10- and 22-hr incubation periods after the removal of free flagellates
were significantly greater than those effected by MPM which had been mock
treated with medium alone. Similar results were obtained when HBM were
used (Table 2). Two alternative explanations were compatible with these
observations: LF-treated cells could destroy more parasites simply
because they had initially taken up larger numbers of organisms or because
they had been activated. To test these possibilities, we measured the
effect of LF on the cytotoxic capacity of MPM after parasite
internalization. The results revealed that substantial parasite growth
had occurred in the mock-treated MPM over a 72-hr period whereas the
LF-treated MPM were able not only to contain growth but also to destroy

many organisms (Table 4).

Mechanism of parasite killing, Killing of the intracellular, amastigote

92

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g N mo.

hzwzh<wmhmma m4 amhm< 2a: mo mbzmhzou mh~m<m<m mzh zH mzomk<~m<> u m m4m<h

93

Footnotes to Table 3

The MPM cultures were incubated with 10 pg LF/ml for 2 h, washed and
then incubated with trypomastigotes for 2 h. After removing the free
organisms, some cultures were fixed (0 h) and the rest received fresh
DM£M+FBS and were further incubated for the indicated periods of

time.

See footnote § under Table 1.

94

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Figure 1

96

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o3.... ”...... ...... ... H .5 N. 3&5...
.... . .... H ...... .8. ....N u o... .... ..
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www.macoa .oz gu.3 2a: a mo space. acmEummch

 

 

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97

Footnotes to Table 4

MPM monolayers were incubated with the parasites for 2 h and washed
to remove the non-bound organisms. whereas some cultures were
immediately terminated by fixation (top row of values), the remainder
received either DEM+FBS or the same medium containing 10 pg LF/ml,
and were further incubated for the indicated periods of time before
termination.

See footnote § under Table 1.

A negative ZR represents an increase in the parasite load of MPM.

98

form of T. cruzi by unstimulated MPM was previously found to be mediated
by H202 (Villalta & Kierszenbaum, 1984a). To find out if LF-treated

MPM killed the blood forms of this parasite by a similar mechanism, MPM
were first pretreated with 10 ug LF/ml for 24 hr, then incubated with the
flagellates for 2 hr, and later on washed with and incubated in fresh
medium for an additional 24 hr, having catalase -a scavenger of H202-
present or absent during these steps. As can be seen in Table 5,
catalase, but not heat-inactivated catalase, markedly inhibited parasite
killing by LF-stimulated MPM. Catalase and heated catalase had no effect

of their own on parasite internalization by MPM whether or not these cells

had been treated with LF.

mama axe: mum omao.n mmuccuoo. Lo.

 

 

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.... H N... .... ..H .... m... n .... .... H ...m. 3238
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.8. ...N u .... .8. .... u N... .8. ....U n ...N 8.. ...N .... N..N 2...... ... .5
.... u ...... ...N H ...... ...... H ..m. ...N ..N m.N. 8.12m...

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8332.... 6.. ...... ...... u 8:88.. .2. 5... ...... a «...... ...
3 mm s o unusucw.»

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i

 

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2;: ouh<mahuu4 no >h~u<a<u mzk zo mm<4<h<u mo wozwmwma mxh mo mhumumw u m m4m<h

T.§

100

Footnotes to Table 5

MPM monolayers were treated with LF in the presence or absence
of catalase or heat-inactivated catalase (HI-catalase) for 24 h,
washed and incubated further with T. cruzi for 2 h in the
presence or absence of catalase or HI-catalase. After removing
the non-bound organisms, the cultures were either fixed
immediately (0 h) or incubated for an additional 22 h with fresh

medium containing catalase or HI-catalase before termination.

See footnotes under Table 1.

101

DISCUSSION

The conditions inducing increased trypomastigote uptake varied
depending on whether MPM or HBM were used. MPM had to be pretreated with
LF for at least 24 hr for the phenomenon to be seen (Table 1) whereas HBM
required only a Z-hr pretreatmnet (Table 2). Van Snick and Masson (1976)
determined the average number of LF receptors per MPM to be 2 X 107 per
cell and Bennett and Davis (1981) found the corresponding value for HBM to
be 2 X 108; the presence of a greater number of LF receptors on the HBM
could explain why these cells responded faster than MPM to LF in terms of
increased parasite uptake.

The number of receptors for LF on the trypomastigotes, must be
relatively small, if any, because indirect immunofluorescence tests to
detect bound LF on these organisms were systematically negative. The lack
of receptors for LP on trypomastigotes and the fact that pretreatment of
this parasite form did not alter its uptake by untreated MPM rendered
unlikely the possibility that LF could have increased parasite uptake by
establishing a molecular bridge between the two cells.

Hhether L? was present during the Z-hr period of MPH-T. cruzi
interaction or the MPM were pretreated with LF for 2 hr, the percentages
of MPM with parasites did not vary significantly during the 70-hr
observation period (Fig. l and Table 3). Instead, the number of parasites
per 100 LF-treated MPM was significantly reduced during the same period of
time. These findings, viewed in the light of the concomitant increase in
the parasite load of mock-treated MPM, indicated that LF-treated MPH had

an increased capacity to kill T. cruzi trypomastigotes. Indicating that

102

the reduction in parasite load fostered by LF involved parasite killing
was the ability of catalase to inhibit this effect (Table 5). This enzyme
has been shown to inhibit trypomastigote destruction by MPM stimulated by
interferon gamma (Hirth, Kierszenbaum, Sonnenfeld & Zlotnik, 1985). The
development of enhanced cytotoxicity by LF-treated MPM was time-dependent,
requiring preincubation with 10 pg LF/ml for a period of 12 to 24 hr
before being detectable (Table 1). However, HBM developed a similar
capacity after only 2-hr of pretreatment with the same concentration of LF
(Table 2). This kinetic difference might also be a function of the
density of LF receptors on the surface of phagocytic cells alluded to
above.

When the MPM were pretreated with LF for 2 hr, a reduction in their
parasite load was seen 70 hr after removal of the free parasites (Table
3). However, this effect occurred much earlier (i.e., after 10 hr) when
the MPM were pretreated with LF for 24 hr (Table 1). Hhile these results
stress the stimulatory effect of LF on MPM cytotoxicity, they do not
provide a definitive explanation for the noted kinetic difference.

Trypomastigotes were also destroyed at faster rates when MPM which
had already ingested the flagellates were treated with LF (Table 4). This
finding reinforces that LF increases the cytotoxic capacity of MPH and
indicates that enhanced killing was not necessarily dependent on the
initial uptake of a larger number of organisms. The involvement of
H202 in parasite killing by LF-treated MPM was denoted by the
inhibitory effect of catalase (Table 5). The results, however, do not
clarify whether H202 was directly toxic for the parasite or acted

indirectly, via production of other toxic 02 reduction intermediates.

103

Of interest in this context is that H202 is also involved in
trypomastigote killing by MPM when these cells are stimulated with
interferon gamma (Hirth et_al;, 1985). Thus, H202 would play a role
in T. cruzi destruction within MPM following activation by different
stimuli.

Unlike amastigotes (Lima & Kierszenbaum, 1985), trypomastigotes did
not bind LF. Furthermore, the macrophages required a longer (24 hr)
pretreatment with LP to display enhanced uptake and killing of
trypomastigotes than to achieve similar effects with amastigotes (1 hr).
These differences, for which we can not provide an explanation at the
present time, are possibly related to the distinct biological features of
these two life cycle stages of T. cruzi.

The noted activities of LF might be related to the level of iron
saturation, its cationic property, or to both. Finally, it is noteworthy
that the concentrations of LF found to enhance phagocyte interaction with
T. cruzi are within the range found in the plasma of patients with
inflammatory conditions (Hansen, Karle, Andersen, Malmquist & Hoff, 1976;
Zenian & Kierszenbaum, 1982; Lima & Kierszenbaum, 1985) similar to those
occurring in T. cruzi infection and might be involved in host defense.

These possibilities deserve further study.

MXNOHLEDGEFENTS

This work was supported by Research Grants AI 14848 and AI 17041

from the United States Public Health Service.

104

REFERENCES

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macrophage menbrane components in the phagocytosis of bloodstream
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ANDRADE, Z. A. & ANDRADE, S. G. 1979. Patologia. In: Trypanosoma

 

cruzi e Doenca de Chagas, pp. 199-248 (Edited by Brener, Z. &
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BENNETT, R. M. & DAVIS, J. 1981. Lactoferrin binding to human peripheral
blood cells: an interaction with a B- enriched population of
lymphocytes and a subpopulation of adherent mononuclear cells.
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BUDZKD, D. B. & KIERSZENBAUM, F. 1974. Isolation of Trypanosoma cruzi

 

from blood. Journal of Parasitology_60:1037-1038. _
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Isolation of the intracellular stage of Trypanosoma cruzi and its

 

interaction with mouse macrophages in vitro. Infection and

 

Immunity 33:546-554.

connsux, H. C. & KIERSZENBAUH, F. 1984. Modulation of macrophage
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CONNEUN, H. C. & KIERSZENBAUM, F. 1985. Increased host cell-
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of the parasite surface. Molecular and Biochemical Parasitology
17:191-202.

105

DE ALMEIDA MARIA, T., ALCANTARA, A. & BRENER, Z. 1982. Ultrastructural
studies on the in vitro interaction of Trypanosoma cruzi
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HANSEN, N. E., KARLE, H., ANDERSEN, V., MALQUIST, J. & HOFF, G. E. 1976.

 

Neutrophilic granulocytes in acute bacterial infection. Sequential
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Experimmental Immunology_26:463-468.

 

KIERSZENBAUM, F. 1979. Antibody-dependent killing of bloodstream forms

of Trypanosoma cruzi by human peripheral blood leukocytes.

 

American Journal of Tropical Medicine and Hygiene 28:965-968.
KIERSZENBAUM, F. & HAYES, M. M. 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.

KIERSZENBAUM, F., KNECHT, E., BUDZKO, D. B. I: PIZZIMENTI, M. C. 1974.
Phagocytosis: a defense mechanism against infection with
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KIERSZENBAUM, F., VILLALTA, F. & TAI, P.-C. 1986. Role of inflammatory
cells in Chagas' disease. III. Kinetics of human eosinophil
activation upon interaction with parasites (Trypanosoma cruzi).

Journal of Immunology 136:662-666.

 

 

LIMA, M. F. B KIERSZENBAUM, F. 1985. Lactoferrin effects on phagocytic
cell function. I. Increased uptake and killing of an intracellular

parasite by murine macrophages and human monocytes. Journal of

 

Immunology 134:4176-4183.

106

NATHAN, C., NOGUEIRA, N., JUNAGBHANICH, C., ELLIS, J. & CDHN, Z. 1979.
Activation of macrophages in vivo and in vitro. Correlation between
hydrogen peroxide release and killing of Trypanosoma cruzi.

Journal of Experimental Medicine 149:1056-1068.

0KABE, K., KIPNIS, T. L., CALICH, V. L. G. & DIAS DA SILVA, N. 1980.
Cell-mediated cytotoxicity to Trypanosoma cruzi. I.
Antibody-dependent cell-mediated cytotoxicity to trypomastigote
bloodstream forms.Clinical Immunology and Immunopathology
16:344-353.

PRYSNANSKY, K. B., MACRAE, E. K., SPITZNAGEL, J. K. & CO0NEY, M. H.
1979. Early degranulation of human neutrophils: immunocytochemical
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ROMANA, C. 1963. In: Enfermedad de Cha as, pp. 97-146 (Edited by
Romaha,C.) Lopez Libreros Editores, Buenos Aires.

SCOTT, M. T. & SNARY, D. 1982. American trypanosomiasis
(Chagas'disease).ln: Immunology of Parasitic Infections, pp.
261-298 (edited by Cohen, S. & Harren, K. S.) Blackwell Scientific
Publications, Oxford.

SPITZNAGEL, J. K. 1984. Nonoxidative antimicrobial reactions of
leukocytes.ln: Regulation of Leukocyte Function, pp. 283-343
(Edited by Snyderman,R.) Plenum Press, New York.

VAN SNICK, J. R. 8: MASSDN, P. L. 1976. The binding of human lactoferrin
to mouse peritoneal cells. Journal of Experimental Medicine

144:1568-1580.

107

VILLALTA, F. & KIERSZENBAUM, F. 1983. Role of polymorphonuclear cells
in Chagas' disease. I. Uptake and mechanisms of destruction

of intracellular (amastigote) forms of Tgypanosoma cruzi by

 

human neutrophils. Journal of Immunology 131:1504-1510.

 

VILLALTA, F. & KIERSZENBAUM, F. 1983a. Role of cell surface

mannoseresidues in host cell invasion by Trypanosoma cruzi.

 

Biochimica et Biophysica Acta 736:39-44.
VILLALTA, F. & KIERSZENBAUM, F. 1984. Role of inflammatory cells in
Chagas'disease. I. Uptake and mechanisms of destruction of

intracellular(amastigote) forms of Trypanosoma cruzi by human

 

eosinophils. Journal of Immunology_132:2053-2058.
VILLALTA, F. & KIERSZENBAUM, F. 1984a. Role of inflammatory cells in
Chagas' disease. II. Interactions of mouse macrophages and

human monocytes with intracellular forms of Trypanosoma cruzi:

 

uptake and mechanism of destruction. Journal of Immunology
VILLALTA, F. & KIERSZENBAUM, F. 1986. Effects of human

 

colony-stimulating factor on the uptake and destruction of a
pathogenic parasite(Trypanosoma cruzi) by human neutrophils.
Journal of Immunology 137:1703-1707.

VILLALTA, F. & LEON, N. 1979. Effect of purification by DEAE-cellulose

 

 

on infectivity of Trypanosoma cruzi blood forms.

 

Journal of Parasitology 65:188-189.

 

HIRTH, J. J. & KIERSZENBAUM, F. 1982. Inhibitory action of elevated

levels of adenosine-B':5' cyclic monophOSphate on phagocytosis:

Effects on macrophage-Trypanosoma cruzi interaction. Journal of

 

Immunology 129:2759-2762.

108

HIRTH, J. J. & KIERSZENBAUM, F. 1983. Modulatory effect of
guanosine-3':5'cyclic monophOSphate on macrophage susceptibility to
Trypanosoma cruzi infection. Journal of Immunology_

121:3028-3031.
HIRTH, J. J. & KIERSZENBAUM, F. 1984. Fibronectin enhances

 

macrophage association with invasive forms of Trypanosoma cruzi.

 

Journal ofImmunology 133:460-464.

 

NIRTH, J. J., KIERSZENBAUM, F., SDNNENFELD, G & ZLOTNIK, A. 1985.
Enhancing effects of gamma interferon on phagocytic cell

association with and killing of Trypanosoma cruzi. Infection

 

and Immunity 49:61-66.

 

NOLACH, B., COATES, T. 0., HUGLI, T. E., BAEHNER, R. L. & BDXER, L. A.
1984. Plasma lactoferrin reflects granulocyte activation in burn
patients. Journal of Clinical and Laboratory Medicine
103:284-293.

ZENIAN, A. & KIERSZENBAUM, F. 1982. Inhibition of
macrophage-Trypanosoma cruzi interaction by concanavalin A and
differential binding of bloodstream and culture forms to the

macrophage surface. Journal of Parasitology 68:408-415.

 

109

CHAPTER III

LACTOFERRIN EFFECTS ON PHAGOCYTIC CELL FUNCTION.
DISTINCT ROLES IN MODULATION 0F MACROPHAGE INTERACTION WITH
A PARASITE (Trypanosoma cruzi)

 

110

ABSTRACT

The present studies on the role of iron in the ability of
lactoferrin (LF) -a neutrophil glycoprotein whose levels in body fluids
increase in inflammatory conditions- to stimulate macrophage functions
unveiled two distinct modes of action involved in modifying uptake and
killing. The presence of iron was not a requirement for LF to enhance the
capacity of mouse peritoneal macrophages (MPM) to take up Trypanosoma
gyg51_amastigotes (AMA) or latex particles. In contrast, iron was
required to be in the molecule in order to stimulate AMA killing by the
MPM. Thus, ApoLF was incapable and LF lost its ability to enhance AMA
killing in the presence of the iron chelator deferoxamine. Shnilar
results were obtained by using diethylenetriaminepentaacetic acid, an iron
chelator which is not incorporated into cells, suggesting that iron had to
be a part of the LF molecule while LF was on the MPM membrane. Moreover,
ApoLF did increase MPM killing of AMA when ferric ions were restored to
the molecule. Such restoration was demonstrable also when iron was added
to the culture medium after MPM treatment with, and removal of the
non-bound, ApoLF. Interestingly, ferrous and ferric ions were equally
effective in restoring activity to ApoLF and, though to a lesser extent,
also zinc. Transferrin did not alter the capacity of MPM to either take
up or kill the AMA, suggesting that the noted LF effects were probably
specific for the latter protein. Immunofluorescence studies revealed that
ApoLF and LF at either 20 or 100% iron saturation bound to approximately
the same proportion of MPM, and that the fluorescence intensity of

positive cells was comparable in all three cases. Thus, the lack of

111

effect of ApoLF on MPM killing is unlikely to be due to ApoLF not binding
to MPM. Killing of internalized AMA by LF-treated MPM, known to be

mediated at least in part by H202, OE'and 1

02, was found in this

work to involve also OH' Nhile providing information concerning the
requirements for LF to enhance macrophage killing of T. cruzi, these
results also evidence a clear separation of the uptake and killing

functions of macrophages which depends on the activating stimulus.

112

INTRODUCTION

Lactoferrin (LF), is a typical example of a secretion product of
an inflammatory-type cell, the neutrophil (1; reviewed in reference 2),
which can modulate the functions of another inflammatory cell. For
example, LF has been reported to inhibit the production of colony-
stimulating factor by human monocyte-derived (3) and mouse (4, 5)
macrophages, and to increase monocyte tumoricidal activity (6). He
previously reported that treatment with lactoferrin (LF) increases the
capacity of mouse resident peritoneal macrophages (MPM) to interact with

amastigote forms of Trypanosoma cruzi (AMA), the causative agent of

 

Chagas' disease (7). In this case, the LF effects translated into greater
uptake of this parasite, followed by killing at a significantly faster
than normal rate. This model system of macrophage interaction with a
microorganism was used in the present work to explore the molecular
requirements for LE to evoke the above-mentioned effects. LP is an
iron-binding protein (reviewed in references 8 and 9) and the presence or
absence of iron ions in the molecule has been shown to be an important
factor in determining its ability to inhibit colony-stimulating factor
production by human macrophages (3, 5). For these reasons, we examined
whether iron was a requirement for LP to enhance the uptake and killing of
T. cruzi by MPM. The results presented in this paper established that

the presence or absence of iron in the LF molecule affords a clearcut
distinction between these two macrophage functions that is emphasized by

different molecular requirements.

113

MATERIALS AND METHODS

Parasites. Tulahuén strain T. cruzi amastigotes were
prepared as described in detail previously (10). Parasite suspensions at
2.5 x 107 organisms/ml were prepared in minimal essential medium with
Hank's salts containing 100 IU penicillin and 100 pg streptomycin per ml
(FEM) plus 1% BSA (NEM+BSA). All of the suspensions used in this work
consisted exclusively of amastigotes (>991 viable).

Reagents. Human LF was purchased from Sigma Chemical Co. (St.
Louis, MO) and was verified to be 20% iron saturated as described by
Ambruso and Johnston (11). Iron-free LF (ApoLF) was prepared by first
incubating a mixture containing 500 pg/ml LF and 0.25M deferoxamine
(Ciba-Geigy, Sumnit, NJ) in phosphate-buffered saline pH 7.2 (PBS) at
37°C for 1 hr; deferoxamine was removed by dialysis at 4’0 vs. PBS.
This material was determined to be iron-free. LF was saturated with
ferric ions by incubating a mixture containing 1 mg/ml LF in PBS with lmM
ferric citrate at room temperature for 24 hr. Excess iron ions were
removed by dialysis vs. PBS (4°C, 24 hr, three changes of PBS). The
solutions of these proteins were aliquoted and stored at -20°C until
used. Human Transferrin at 0% and 100% iron saturation were purchased
from Calbiochem (La Jolla, CA). All necessary dilutions of these reagents
were made in MEM+BSA.

Mouse peritoneal macrophages (MPM). The methods to obtain MPM
from Crl:CD-1(ICR)BR Swiss mice (Charles River Laboratory, Portage, MI)

and to prepare monolayers of these cells have been described elsewhere(7).

114

The cultures routinely consisted of >981 nonspecific-esterase positive
cells with typical macrophage morphology.

Pretreatment of MPM with LF or ApoLF. Monolayers of MPM were
incubated at 37°C for 1 hr with 0.2 ml of MEM+BSA alone or containing 10
pg/ml of Apolactoferrin (ApoLF) or LP at 20% or 100% iron saturation.
The monolayers were then washed three times with MEM and used in the
assays to determine macrophage-parasite interactions (see below).

Determination of MPM association with AMA. All experimental and
control conditions were tested in triplicate. MPM cultures treated with
either LP or MEM+BSA received 0.1 ml of AMA suspension plus 0.2 ml of
MEM+BSA and were incubated at 37°C in a 5% COZ-in-air atmosphere for 2
hr. After removing the free organisms by five washings with MEM, the
cultures were fixed with absolute methanol and stained with Giemsa. At
least 200 cells were examined microscopically (X1000) on each culture.
The number of parasites associated with these cells and the number of MPM
with and and without parasites were recorded and used to calculate the
mean percentage of MPM with parasites and the average number of organisms
per 100 MPM.

Determination of MPM killing of AMA. A replicate set of
cultures initiated as described above was further incubated -after
renoving the non-bound AMA- with 0.3 ml of MEM supplemented with 10%
heat-inactivated (56°C, 1 hr) FBS dialyzed vs. PBS (4°C, 24 hr)
(iEM+dFBS) at 37°C (5% C02) for an additional 6 hr. Termination and
processing of these cultures were as described above.

In experiments designed to test the iron dependency of the

observed LF effects on MPM function, MPM were simultaneously incubated

115

with LF and various concentrations of deferoxamine (see Results) at
37°C for 1 hr. These reagents were washed off with MEM prior to exposing
the MPM to AMA.

To investigate the mechanism(s) underlying our observations, a
protocol was used in which deferoxamine, diethylenetriaminepentaacetic
acid (DTPA), urea, thiourea or oc-ketobutyric acid was present in the
medium from 2 hr after placing the MPM into the culture chambers until
termination of the experiment.

In experiments designed to test the effect of cation restoration
of the LF effects on NEW association with and killing of AMA, the relevant
cation (see Results) was added to the culture medium immediately after
M84 pretreatment with ApoLF and the cultures were further incubated for 1
hr. After washing three times with MEM, the parasites were added. The
rest of the experiment proceeded as described above for determination of
both MPM association with and killing of AMA.

Indirect immunofluorescence assay for LF. The method to
establish LF binding to both MPM and AMA has been described (7).

Latex bead uptake by ApoLF- or LF-treated and mock-treated MPM.
Cultures of MPM were incubated at 37°C (5% C02) for 1 hr with ApoLF or
with LF at 20% or 100% iron saturation and washed three times with MEM
before receiving 0.3 ml of latex bead suspension (8.3 X 106 beads/ml
MEM+BSA; average bead diameter 1 pm, Sigma). After further incubation
for 2 hr and removal of the free heads, the cultures were fixed with
methanol, stained with Giemsa and examined microscopically (X1000) to
establish the percentage of MPM with latex beads and the number of beads
per 100 MPM.

116

Presentation of results and statistics. All results are
expressed as the mean :_1 5.0. Differences were considered to be
significant if P50.05 as determined by the Mann-Whitney “U“ test. The
sets of results presented in the tables are typically representative of

two to three separate repeat experiments.

117

RESULTS
Effects of iron content on the capacity of LF to enhance MPM

association with or killing of AMA. In previous work, we showed that LF

 

at 20% iron saturation enhanced the capacity of MPM to associate with and

kill T. cruzi AMA (7). In the present work, this effect was reproduced

and, furthermore, found to be also induced by LF at 100% iron saturation I
(Table 1). However, iron removal from the molecule prior to its use in E‘
the treatment of MPM virtually abrogated the enhanced killing effect but (#1
failed to alter the capacity of these cells to associate with AMA. ApoLF A I
recovered its capacity to enhance parasite killing afteg iron replacement,

achieved bylnixing ApoLF with ferric citrate (excess iron ions were

removed by dialysis). Ferric ions alone, in the absence of ApoLF, had no

detectable consequence on the extent of either MPM-parasite association or

killing. To confirm the iron requirement for LE to enhance AMA killing,

we used an alternative approach in which the MPM were incubated with LP in

the presence of the iron chelator deferoxamine and then exposed to the

parasites. A representative set of results is presented in Table 11,

showing that the extent of parasite killing by LF-treated MPM was reduced

from 73% to 44% and 10% in the presence of 0.5mM and SmM deferoxamine,

respectively. The lowest concentration of deferoxamine tested, 0.05mM,

was ineffective in curtailing the stimulatory effect of LE on parasite

destruction. Deferoxamine alone had no detectable effect on either MPM

association with and killing of AMA at any of the tested concentrations.

In some experiments, we used DTPA, an iron-chelating agent which, unlike

deferoxamine, can not enter cells (12). As shown in Table III, DTPA was

as effective in inhibiting the LF-induced enhancement of AMA killing as

mama .xm: mom mmew.a mucocuoo. Lo.

 

 

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. .... .8..m. 8...“...ON 9....“ 8.88 ..oa< um.amc.-m.e...u u.ctm.
mm N...“ 8.88 no.m.fi.o..m ..ca<
.o.. ...... m...u_o.ON .8...“ 8... =o...c:.~m cat. moo. ...

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... n ...N ...N H 8.8 «8:82

c: m L: N acosueoeuwga

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nu.) =o.uo.uomme an: mucmscm o» J. .o a»...ae mg. no unmacou cog. .o muummmm

 

a w—nop

119

Footnotes to Table I

aCultures of MPM were incubated with the solution of the
indicated material(s) at 37°C for 1 hr, washed with MEM and then
incubated with AMA for 2 hr. Half of the cultures were then terminated by
fixation whereas the other half received MEM+dFBS and was incubated for an
additional 6 hr. ApoLF or LF were used at 10 pg/ml.

bThe difference between this value and the corresponding control
was statistically significant (P50.05). Values in brackets represent
the actual decrease in the average number of parasites per 100 MPM.
Values in parentheses represent the percentage of reduction in parasite
contents occurring over the 6-hr time interval. Values in brackets and

parentheses are shown only where the difference was statistically

significant with respect to the corresponding 2-hr value.

 

120

.. m.aa» o. muuocuoc. mam

....

 

 

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8... H ...N ..n H ...N =5... 2.838....

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c... H ...... A...N H ..8 2.53888 :8 + ..

.... Hm..N. ..N.H ...m am...H ..mm a=.s~xoca.oe sam.c + m.
.88. .m.8m. m.o.H m... .....H a... «c.2exoc~.au samo.o + ..
8.. 8.... m.N H ...... ...... H ...... ....
N.. H ....N .... H ...m «8+5:

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couue 282 cc. Ema <z< .o .oz mmoggocumz

 

e.~=cu .h .o

 

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.. mpnmh

121

.l Flirty.

emen axe: mmem.a mmuoeuoo. so.

 

 

 

..N.H 8.8. ..N.H o... m=.e~xoca.ma

8.o.H o.mm a..N_H_8.eN a=.smxotm.ae 2582+ ..

..._H_8.NN m.o.H 8... «..o

..N H.8..m n..m_H_m..m «... as...o.+ ..

.... ....N. e.o.H 8.8 A.....H 8.88 m.
...m H ...N 8.. H ...... «8......

L; m L: N acoEueocumcn

cm..e 2;: 00. can <z< .8 .oz omezaocuez

u.~=cu .h

 

.OTmc..... 2.: auemcem 0. m. .o ......a as. :0 a:.e..o.a.au to «..a .o 8.88...
... 28.

122

Footnotes to table III

aHhen designed to be present, DTPA or deferoxamine was added to
the culture medium 2 hr after placing the MPM in the culture chambers and
remained present until the end of the experiment, including washings.
Otherwise, the protocol was as described in the footnote to Table I.

bSee footnote to Table I.

123

deferoxamine. It is noteworthy that, under the tested conditions, neither
deferoxamine nor DTPA had any detectable effect on MPM or AMA viability as
determined by trypan-blue exclusion and vibratile in situ motion,
reSpectively. Nhen results obtained in repeat experiments were compared,
the extents of the noted effects sometimes varied. However, the effects
themselves were always readily reproducible and statistically significant.
He considered the possibility that the absence of iron in the LF

molecule might have affected its ability to be bound by MPM and conducted

 

several types of experiments to test it. When MPM were incubated with *
solutions containing the same concentrations of ApoLF, LF at 20% iron
saturation and LF at 100% iron saturation, the percentages of cells
binding these proteins, determined by immunofluorescence, were 76 :_4,

84 :_6 and 86 :_8 percent, respectively; the control value obtained

with MPM incubated with MEM+BSA alone was 0 :_0 percent. The intensity

of the fluorescence of the positive MPM was comparable in all three cases.
Moreover, MPM pretreated with ApoLF, LF at 20% iron saturation and LF at
100% iron saturation increased the capacity of MPM to ingest latex beads
similarly (from a control level of 57 1.3 percent to 125 :_11, 107 :_

11 and 135 :_2 percent, respectively). This had also been the case with
AMA uptake (Table I). In an alternative approach, we incubated the MPM
first with ApoLF and, after washing the cells exhaustively, incorporated
ferric ions into the system in an amount sufficient to have saturated the
initial amount of LF. The result was enhanced AMA killing (Table IV).
Iron added to MPM cultures which had been mock-treated with MEM+BSA alone

had no detectable consequence.

The latter protocol was used to find out also whether other

omen uxmc mom mmuocuoo. so.

 

124

 

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..m H 8.8 .... H ...N t... z. ..8 «8+5:
... H 8.8 ...N H 8.8 1... EN. «8+5:
m. H 8.8 8.. H ...N 1+... .88. «8+5...
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.88. .e.mN. o.m.H o.ON n..o.H 8.m« +++m. gam.N. ..oa«
o... H 8.8 A.... H 8.... «8+5... :2:
.mo. .N.om. m.«.H.m... an...H m..« «88+25: =o..ec=.em :oc. use. 8.
m... H ...8 m... H «.8 «8+5: «8+5...
2. 8 c. N m .888 « aaum

588.8 :8: oo. .88 <z< .o .oz acme.amcumcn wmognocuaz

 

 

e.N=cu .. .o.me..... zaz aueegca 0. ..oa« .8 8......

 

ozu acoamac a» use. uwumsu new u=.~ «maocgm. .u.ccm. .o xu.uumeu m>.ue.wm

>~ mpnoh

125

Footnotes to Table IV

aStep A: cultures of MPM were incubated with the solutions of
the indicated materials at 37°C for 1 hr and washed with MEM. Step 8:
the cultures were further incubated with the indicated solutions at 37°C
for 1 hr, washed and then incubated with AMA for 2 hr. Half of the
cultures‘were terminated by fixation whereas the other half received
MEM+dFBS and was incubated for an additional 6 hr. ApoLF or LP were used
at 10 09/ml.

bSee footnote to Table I.

126

cations previously reported to bind LF (13, 14) could also restore the
capacity of ApoLF to induce greater AMA killing. As shown in Table IV,
ferrous but not cupric cations afforded total restoration, and zinc had
only a partial effect.

To test whether the effects of LF on MPM interaction with I;
95251 AMA were characteristic of this protein or were shared by another
iron-binding protein, parallel experiments were performed using
iron-saturated LF, ApoLF, transferrin and apotransferrin. Neither
transferrin, tested at concentrations ranging from 1 to 10,000 pg/ml, nor
1,000 pg apotransferrin per ml could enhance MPM association with or
killing of T. cruzi whereas the effects of apoLF on MPM-AMA association
and those of iron-saturated LF on both association and killing were
readily seen (Table V). In related experiments we tried to restore the
ability of ApoLF to enhance killing of T. cruzi AMA by pretreating the
MPM with a 10pg/ml solution of this protein in the presence of 1000pglml
iron saturated transferrin and later exposing these macrophages to the
parasites. Hhereas ApoLF, ApoLF in the presence of transferrin or
transferrin itself were not able to stimulate killing of AMA by MPM, this
ability was readily demonstrated by a solution of 100% iron saturated LF
(data not shown).

Effects of scavengers of hydroxyl radical on AMA killing by

LF-treated MPM. 'He have previously shown the involvement of at least
1

 

H202, 05 and

MPM (7). There have been reports that LF stimulates OH' production by

02 in the killing of T. cruzi AMA by LF-treated

neutrophils (11) and iron ions have been shown to play a role in the

formation of OH' radicals via the Haber-Heiss reaction (15).

127

mung uxm: mum mmoo.a mmuocuoo. co.

 

 

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N.. H ...: ... H ...... .....9. 88.. £28.22.
«... H 8.8 .... H ...N. 8.9. 8. £28.22.
N... H ...... ..N H .... .....9. ... 228.22.
...m H ...8 ...« H ...... .....9. . £28.28.
.... H ..8 N.N H ...8 «8+5: N
.... H ....N ...... H ...N .....9. ..8.. £28.22.
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.8. 8.3 N.. H 8... .... H ...8 2.9. ... .8223: 8... .8. ....
...... H ...... ..m H m... 8.8... ......2.«
..m H ”...N ...N H ...N «8+5: .
.... 8 .... N .2.
qu.a ta: co. gun <z< .0 .oz “coauamgawcn wmmgnogumz .nxm

 

 

o<z< .... .o :u.: muo.uommm o» in: .o Nu.ueaeu mg. co uu~..m c...~.m:e.u .o xue.

> mpnoh

128

Footnotes to Table V

aCultures of MPM were incubated with the indicated solutions at
37°C for 1 hr, washed with MEM and then incubated with AMA for 2 hr.
Half of the cultures were terminated by fixation whereas the other half
received MEM+dFBS and was incubated for an additional 6 hr.
Transferrin was 100% iron saturated.

bSee footnote to Table I.

129

Therefore, we looked into the possible involvement of OH’ in AMA killing
in our assay system. The results indicated that thiourea and oc-ketobu-
tyric acid, both scavengers of OH' radicals (12, 16), significantly
reduced the extent of the enhancement of MPM cytotoxicity induced by.LF
(Table VI). Urea, a thiourea analog lacking this scavenging property, was
ineffective. At the tested concentrations, thiourea, urea and oeketo-
butyric acid did not affect the viability of MPM or the AMA to any

appreciable extent (data not shown).

13D

wmea uxm: mom umoo.a muuocuoo. go.

 

 

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...N H N..... ..N.. H m... 39. 82:8338 :28 + ...

m..Hm8. N..H..... 8.: :58

.... 2.8. ...N H 88 a: H ...8 88 :8. + ..
.... H .8. N.N H 8.... 8.8.5 :28

... H ..8 ...... H 8.8 8.8.5 :28 + ..

.8. 8.8. N8 H 8.... ...... H 8.8 ...
. ...... .+. 8.8 ...N H .8 «8+5:

L: m g: N unusuemguogn

Lou.o 2.: oo. .88 <z< .o .02 mmmgaoguez

 

 

 

eta: uuuougu-.. an m:..... <z< .~=.u .» co m.~u.uu. .axc.cag .o m.mm:m>oum .o maum..m

.> m—nop

131

Footnotes to Table VI

aNhen designed to be present, thiourea, urea or oc-ketobutyric
acid was added to the culture medium 2 hr after placing the MPM in the
culture chambers and kept in it throughout the entire experiment,
including washings. Otherwise, the protocol was aS'described in the
footnote to Table I.

bSee footnote to Table I.

132

DISCUSSION

The observation that iron-containing LF but not ApoLF enhanced the
capacity of MPM to kill T. cruzi, together with the recovery of such
activity afforded by iron restoration to ApoLF, contrasted with the lack
of an iron requirement for this glycoprotein to enhance MPM-parasite
association (i.e., binding and internalization). This difference infers
at least two separate mechanisms for the noted stimulatory effects of LF.
The increased capacity of MPM to take up AMA or latex beads after MPM
incubation with either ApoLF or LF attributed this property to the
glycoprotein itself. Since both ApoLF and LF were found to bind to the
MPM surface and receptors for LP have been shown to be present on the
surface of both MPM (17) and T. cruzi AMA (7), any of these molecules
could have conceivably established a molecular bridge between MPM and AMA,
facilitating parasite uptake in a manner similar to opsonization.

However, ApoLF and LF also stimulated latex bead uptake by MPM, suggesting
that a bridging mechanism was not necessary for MPM to denote a greater
phagocytic capacity. Nevertheless, bridging can not be ruled out as a
contributing mechanism.

The concept that enhanced AMA killing required the presence of
iron in the LF molecule was supported by several lines of evidence.

First, parasite killing was enhanced by iron-containing (whether at 20 or
100% saturation) LF but not by ApoLF. Second, enhanced killing was not
seen when the MPM were treated with iron-containing LF in the presence of
deferoxamine, an iron chelator. The magnitude of this effect was

dependent upon the concentration of deferoxamine and comparable results

133

were obtained by using DTPA, another iron chelator. Third, we were able
to induce killing enhancement with ApoLF after iron restoration. It is
noteworthy that the tested concentrations of deferoxamine or DTPA were not
cytotoxic for the MPM and, therefore, could not have prevented parasite
disposal via killing the host cell.

The presence of DTPA in the culture medium during MPM treatment
with LF prevented the enhancement of AMA killing. Since this chelator can
not enter into living cells (12), these results suggest that DTPA must
acquire iron from the LF molecule in the culture medium or while the
protein is bound to the MPM surface to suppress greater killing activity.
The idea that ApoLF is binding to the MPM surface is supported by the fact
that the ability to enhance MPM killing of AMA was restored to ApoLF by
free iron ions in the culture medium aj£g[_ApoLF treatment of MPM (i.e.,
after the removal of non-bound protein).

Free iron ions did not induce greater parasite killing by MPM
(Tables I and IV), indicating that this effect required the benefit of a
carrier. Yet, a mere ability to bind and transport iron was not sufficient
since the phenomenon could not be induced with transferrin (Table V).

Hypothetically, LF could provide distinct signals to MPM, leading
to greater phagocytic and cytotoxic capacities. Binding of the
glycoprotein, regardless of the presence of iron, would increase
phagocytosis whereas iron would also be required to trigger the toxic
mechanism(s). Alternatively, surface-bound LF might initiate the process
that leads to increased killing but iron transported into cell by LF could
be essential for the effector mechanism to be mounted. However, this

possibility was not in keeping with the results of experiments in which

the MPM were simultaneously treated with ApoLF and transferrin

134

[which is known to bind to surface receptors other than those for LF (17,
18) and should have transported iron into the cells], since killing was
not increased under these conditions. 0n the other hand, iron brought in
by transferrin might not have been found where ApoLF could have used it.

The present results do not specify if the presence of the
appropriate cation in the LF molecule causes the molecule to bind to a
specific MPM surface site(s) -different in some manner from that to which
ApoLF binds- enabling it to elicit enhanced cytotoxicity. However, ApoLF
did stimulate AMA killing when zinc was present instead of iron (Table
IV). This finding could be interpreted in terms of a need for LF to be in
a certain conformation (attained best with iron) necessary for the protein
to bind to the proper receptor to be active in promoting killing or the
participation of iron and/or zinc in the cytotoxic mechanism.

Iron plays a role in the generation of oxygen reduction
metabolites (reviewed in reference 15), some of which have been shown to
mediate MPM killing of T. cruzi AMA (7). Internalized LF might_promote
AMA killing within MPM via iron transport. If so, this property would be
unique to LP because transferrin had no detectable effect on AMA killing
by MPM even when used at 100% iron saturation and tested over a
10,000-fold concentration range (Table V). Hhen either thiourea or
oc-ketobutyric acid, both scavengers of OH', were present in the culture
medium, the LF effect on parasite destruction was abrogated, suggesting
that this oxygen reduction metabolite was involved in the effector mech-
anism. This observation adds OH' to the list of oxygen metabolites that
are produced by stimulated MPH and are directly or indirectly toxic for

T. cruzi AMA [which until now included H202, 0; and 102 (7)].

135

and is in keeping with a previous report that LF stimulates OH’

production by neutrophils (11). In our earlier work, we found that
sodium benzoate, a scavenger of 0H°, would not inhibit the stimulatory
effect of LF on AMA killing by MPM (7). The present results, showing that
two other OH' scavengers did inhibit such effect, implicated this

reactive oxygen intermediate in AMA killing by LF-treated MPM but did not
explain the negative results obtained with sodium benzoate.

Ne have found that LF levels are increased in the serum of acutely
infected chagasic mice and that AMA from the spleens of these animals show
intense fluorescence after incubation with fluorescein-labeled rabbit
anti-LF (Lima and Kierszenbaum, unpublished results). This finding and
the fact that LF presented to MPM on the surface of T. cruzi AMA also
enhances phagocytosis and killing (7) indicate that the conditions for the
production of the in vitro effects described in this paper are present

in an infected host.

136
REFERENCES

Masson, P. L., J. F. Heremans, and E. Schonne. 1969.
Lactoferrin, an iron-binding protein in neutrophilic leukocytes.
J.Exp. Med.130:643.

Spitznagel, J. K. 1984. Nonoxidative antimicrobial reactions
of leukocytes. In Regulation of Leukocyte Function. R.
Snyderman. ed.Plenum Publishing Corp., New York. P. 283.
Broxmeyer, H. E., A. Smithyman, R. R. Eger, P. A. Meyers, and M.
de Sousa. 1978. Identification of lactoferrin as the
granulocyte-derived inhibitor of colony-stimulating activity
production. J. Exp.Med. 148:1052.

Broxmeyer, H. E., and E. Platzer. 1984. Lactoferrin acts on
I-A and I-E/C antigen subpopulations of mouse peritoneal
macrophages in the absence of T lymphocytes and other cell types
to inhibit production of granulocyte-macrophage colony stimulatory
factors in vitro. J.Immunol. 133:306.

Broxmeyer, H. E., L. Juliano, A. Haheed, and R. K. Shadduck.
1985. Release from mouse macrophages of acidic isoferritins that
suppress hematopoietic progenitor cells is induced by purified L
cell colony stimulating factor and suppressed by human
lactoferrin. J. Immunol.135:3224.

Nishiya, K., and 0. A. Horwitz. 1982. Contrasting effects of
lactoferrin on human lymphocyte and monocyte natural killer
activity and antibody-dependent cell-mediated cytotoxicity. gg_
Immunol. 129:2519.

10.

11.

12.

13.

14.

137

Lima, M. F., and F. Kierszenbaum. 1985. Lactoferrin effects on
phagocytic cell function. 1. Increased uptake and killing of and
intracellular parasite by murine macrophages and human monocytes.
J. Immunol. 134:4176.

Aisen, P., and A. Leibman. 1972. Lactoferrin and transferrin:

a comparative study. Biochim. Biophys. Acta. 257:314.
Bezkorovainy, A., and R. J. Zschocke. 1974. Structure and
function of transferrins. 1. Physical, chemical, and
iron-binding properties. Arzneim.-Forsch. 24:476.

Villalta, F., and F. Kierszenbaum. 1982. Growth of isolated
amastigotes of Trypanosoma cruzi in cell-free medium. ‘J;
Protozool.29:570.

Ambruso, 0. R., and R. B. Johnston. 1981. Lactoferrin enhances
hydroxyl radical production by human neutrophils, neutrophil
particulate fractions, and an enzymatic generating system. ‘Jy
Clin. Invest. 67:352.

Doroshow, J. H. 1986. Role of hydrogen peroxide and hydroxyl
radical formation in killing of Ehrlich tumor cells by anticancer

quinones. Proc. Natl. Acad. Sci. USA 83:4514.

Ainscough, E. H., A. M. Brodie, and J. E. Plowman. 1982. Zinc

transport by lactoferrin in human milk. Am. J.Clin. Nutr.

33:1314

 

Masson, P. L., and J. F. Heremans. 1968. Metal-combining
properties of human lactoferrin (red milk protein). 1. The
involvement of bicarbonate in the reaction. Eur. J. Biochem.

6:579.

 

15.

16.

17.

18.

138

Klebanoff, S. J. 1982. Oxygen-dependent cytotoxic mechanisms

of phagocytes. Adv. Host Defense Mech. 1:111.

Starke, P. E., and J. L. Farber. 1985. Ferric iron and
superoxide ions are required for the killing of cultured
hepatocytes by hydrogen peroxide. Evidence for the participation
of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss
reaction. J. Biol. Chem. 260:10099.

Van Snick, J. L., and P. L. Masson. 1976. The binding of human

lactoferrin to mouse peritoneal cells. J. Exp. Med. 144:1568.

 

Bennett, R. M., and J. Davis. 1981. Lactoferrin binding to
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mononuclear cells. J.Inmunol. 127:1211.

139

APPENDIX

140

APPENDIX

Lactoferrin is a glycoprotein present in the neutrophil specific
granules (1) and is normally found in plasma in trace amounts (2).
Inflammatory conditions, such as bacterial infections (3,4) and
arthritis (5), are accompanied by elevated concentrations of lacto-
ferrin in plasma. Since inflammation with neutrophil infiltrates is

present in acute Chagas' disease and lactoferrin modulates Trypanosoma

 

£5251 uptake by macrophages and monocytes jg_yjtyg_(6,7,8), we
explored the possible presence of lactoferrin in infected mice and
whether it bound the parasite ig_yjyg,

In the first approach, acute and chronically infected mice were
bled and their sera separated. An indirect immunofluorescence (IFI)
test using cultured amastigote forms of I; Egggi (6) and fluorescein-
conjugated anti-lactoferrin IgG was selected to detect binding of
serum lactoferrin to the parasite. A second approach used spleens
from infected mice as a source of amastigotes, testing for the
presence of lactoferrin jg_§jtg_by direct immunofluorescence using
fluorescein-labeled anti-lactoferrin 196.

Four week old Crl:CD-1(ICR)BR Swiss mice were purchased from
Charles River Laboratories (Portage, MI). These mice were infected
intraperitoneally (i.p.) with 1 x 105 blood trypomastigotes. The mice
were bled at different times post-infection and their sera were

pooled, aliquoted and stored at 20°C until used.

‘5‘!

141

The IFI was done as described previously (6) using culture
amastigotes grown in ML-ISH medium. The single cell suspensions used
in the direct immunofluorescence studies were prepared in a Ten Broeck
tissue grinder in phOSphate buffered saline (PBS) from the spleens of
mice sacrified on day 12 post-infection (p.i.). The suspensions were
kept in an ice bath for one hour, and then used to prepare thin films
on microscope slides, dried and fixed in methanol. The slides were
then covered with normal rabbit IgG for 30 min, washed with PBS and
incubated with FITC-labeled rabbit anti-lactoferrin IgG or
FITC-labeled rabbit anti-human 196 as a control. After washing with
PBS, the slides were examined by fluorescence microscopy.

As shown in Table 1, sera from acutely infected mice (collected
on day 7 p.i., when no parasitemia was detectable and on day 15 p.i.,
when parasitemias attained peak levels) contained LF as detected on
the surface of cultured amastigotes after incubation with sera and
FITC-labeled anti-lactoferrin 196. No fluorescence was detected when
normal mouse serum or serum collected on day 49 p.i. were used.

Figure 1 shows splenic amastigotes displaying fluorescence after
incubation with fluorescein-labeled anti-lactoferrin antibodies.

These observations revealed that lactoferrin was produced during
the acute phase of the disease and reached levels detectable by our
indirect immunofluorescence test, and that lactoferrin can bind
amastigotes in their natural environment. Therefore, the conditions
for lactoferrin binding to amastigotes released from infected cells
are present jg_y1yg, Nhether these conditions lead to increased
parasite uptake and killing by macrophages in the host remains a

subject for further study.

-; "U

142

TABLE I

Increased Levels of LF in Acute Chagasic Sera

 

 

’Amastigotes Percent of LF-
incubated positive

with amastigotes (by IFI)
MEM - 1% BSA 0.: 0.0 *
Normal mouse serum 0.: 0.0

Infected mouse serum,

day 7 p.i. 63.5 i 3.5
Infected mouse serum,

day 15 p.i. 70.4_: 0.5
Infected mouse serum,

day 49 p.i. 0':_0.0
10 ug/ml LF 58.4 i 2.7

 

* Results are expressed as the Mean :_SD

143

Figure 1

Demonstration by direct immunofluorescence of lactoferrin binding to
splenic amastigotes lg situ. Parasites were obtained from spleens of
infected mice at a time of peak parasitemia and stained by fluores-
cein-conjugated anti-lactoferrin IgG.

 

 

144

REFERENCES

Masson, P.L., Heremans, J.F., and Schone. 1969. Lactoferrin, an

iron binding protein in neutrOphilic leukocytes. J. Exp. Med.
130:643-658.

Birgens, H.S. 1985. Lactoferrin in plasma measured by an ELISA
technique: evidence that plasma lactoferrin is an indicator of
neutrophic turnover and bone marrow activity in acute leukemia.
Scand. S. Herematol 34:326-331.

Hansen, N.E., Karle, H., Andersen, V., Malmquist, S., Hoff, G.E.
1976. Neutrophilic granulocytes in acute bacterial infection.
Clin. Exp. Immunol. 26:463-468.

Gutteberg, T.J., Haneberg, B., Jorgensen, T. 1984. The latency
of serum acute phase proteins in meningococcal septicemia, with
special emphasis on lactoferrin. Clin. Chim. Acta 136:173-178.

Bennett, R.M., Skosey, J.L. 1977. Lactoferrin and lysozyme
levels in synovial fluid: differential indices of articular
inflammation and degradation. Arthritis Rheum 20:84-90.

Lima, M.F., and Kierszenbaum, F. 1985. Lactoferrin effects on
phagocytic cell function I. Increased uptake and killing of
intracellular parasite by murine macrophages and human monocytes.
J. Immunol 134: 4176-4183.

Lima, M.F., and Kierszenbaum, F. 198 . Lactoferrin effects on
the interaction of blood forms of Trypanosoma cruzi with
mononuclear cells. Submitted.

 

Lima, M.F., and Kierszenbaum, F. 198 . Lactoferrin effects on
phagocytic cell function 11. Distinct roles in macrophage
interaction with a parasite (Trypanosoma cruzi). Submitted.

 

145

SUMMARY AND CONCLUSIONS

A heavy inflammatory cell infiltrate consisting mainly of
mononuclear cells, neutrophils and eosinophils is frequently seen in
acute chagasic lesions. Secretion products of these cells may play a

role in the interaction between Trypanosoma cruzi and the same or

 

other inflammatory cells. In the course of this project, we focused
on lactoferrin, a glycoprotein secreted by neutrophils, cells present
in acute chagasic lesions. Levels of lactoferrin in the body fluids
are known to be increased during inflammatory conditions such as found
in acute Chagas' disease. This study examines the effects of lacto-
ferrin on the phagocytosis of I;_g§gzi_by macrophages and monocytes,
which are also present in chagasic inflammatory infiltrates.

First, the modulatory effects of lactoferrin on the interaction
between amastigotes or trypomastigotes with human monocytes or mouse
peritoneal macrophages were analyzed. Lactoferrin was found to
stimulate the uptake of amastigotes by either cell in a dose-dependent
manner. This stimulatory effect was maximal at 10 Mg/ml lactoferrin,
a level comparable with that in individuals with inflammatory
conditions. Higher concentrations of lactoferrin were found to be
supraoptimal in that they did not effectively increase host-cell
parasite association.

Pretreatment of either phagocytic cells or parasites with
lactoferrin enhanced their association, suggesting that both

macrophages and parasites bound the protein.

146

Reports by other investigators that macrophages and monocytes
possessed receptors for lactoferrin were readily confirmed in this
work and the presence of receptors for lactoferrin on amastigotes was
demonstrated by binding and competition studies using 125I-labeled
lactoferrin. Since both parasites and phagocytes have receptors for
this protein, lactoferrin might have bridged the two cells and thus
increased their association. However, several lines of evidence
suggested that parasite-cell bridging was not the sole explanation for
this increase. First this enhancement was still demonstrated when
lactoferrin-treated macrophages were incubated with SIC-methyl
mannoside or anti-lactoferrin IgG (agents which block lactoferrin
binding) before addition of the parasites. Second, lactoferrin
enhanced trypomastigote-macrophage association despite the fact that
these forms of the parasite do not bind lactoferrin. Finally, even
the uptake of latex beads by macrophages was enhanced by treating
these cells with lactoferrin. These findings suggest that lactoferrin
has a nonspecific effect on macrophages which stimulates them to
display a greater phagocytic activity, although bridging of parasites
and cells may still contribute to this effect.

I;_g£gzl has been shown to be sensitive to the toxicity of the
intermediates of oxygen metabolism whose production is stimulated
during phagocytosis (reviewed on page 9). Lactoferrin has been shown
to increase the production of some of these intermediates (reviewed on
page 17). Therefore, the increased parasite destruction caused by
lactoferrin could be due to the production of metabolites of the
reduction of oxygen produced by phagocytes upon phagocytosis.

Scavengers of 0; , H202, and 102 were shown to abrogate the

I 147

lactoferrin-stimulated killing of amastigotes, and catalase, a
SCGVEDQEP 0f H202, inhibited trypomastigote killing. Therefore,
lactoferrin may enhance I; 95351 destruction by macrophages via
stimulation of the production of reactive oxygen intermediates.

Because lactoferrin is an iron-binding protein (reviewed on page
12) and iron has been implicated in the partial reduction of oxygen in
phagocytic cells (reviewed on page 9), two separate approaches were
used to investigate the contribution of iron to the lactoferrin
effects. In the first, iron was removed from the protein by

incubation with deferoxamine (an iron chelator). In the second I

 

approach, macrophages were pretreated with lactoferrin in the presence
of deferoxamine and, after removal of both, exposed to amastigotes.

In both cases, it was found that macrophages exposed to Fe-depleted
lactoferrin were unable to kill the amastigotes. However, the cells
retained their ability to phagocytize larger numbers of parasites than
mock-treated controls.

The importance of iron in lactoferrin-mediated killing was
confirmed by the restoration of this effect after incubation of
apolactoferrin with ferric citrate. Lactoferrin preparations at 20
and 100% Fe-saturation were similarly active in stimulating parasite
killing by macrophages.

The inability of Fe-depleted lactoferrin to stimulate parasite
killing was unlikely to be due to a diminished ability of macrophages
to bind the glycoprotein since all lactoferrin preparations,
regardless of iron content, were found to bind these cells to the same

BXtEflt o

148

The role of iron in the lactoferrin-stimulated killing of I;
gygzi_by macrophages was emphasized by the fact that while
apolactoferrin, by itself, could not induce this effect, it could do
so when its iron content was restored either before or after binding
to the host cell. Interestingly, both ferric and ferrous ions were
similarly effective. Addition of zinc instead of iron ions restored
only 50% of the killing level, and cupric ions were totally
ineffective.

The fact that Fe-saturated transferrin could not mimic the
effects of lactoferrin argues against the possibility that lactoferrin
stimulates killing of amastigotes by merely raising the intracellular
iron content of the macrophages.

Next, we set out to explore the mechanism involved in the
iron-dependent lactoferrin stimulation of parasite killing by
macrophages. It should be borne in mind that our previous work had
shown that this killing involved metabolites of the partial reduction
of oxygen. Other investigations have reported that the production of
0H° and 102 is dependent upon the presence of iron. The possible
involvement of iron and these metabolites in parasite killing of
lactoferrin—treated macrophages was studied by using iron chelators
or scavengers of OH' and 102. Both were found to abrogate parasite
killing to significant extents.

In summary, this research has shown that: a) lactoferrin can
stimulate the phagocytic and cytotoxic properties of human monocytes
and mouse peritoneal macrophages, b) some of the ensuing effects are
dependent upon the presence of iron in the molecule, and c) reactive

oxygen intermediates mediate the destruction of I;_cruzi by

149

macrophages. Moreover, results described in the Appendix section
point to the fact that lactoferrin is found in increased amounts in an

jg_vivo infection with I; cruzi and that amastigotes bind to the

 

protein j__situ. These studies forward the notion that a product of
an inflammatory cell modulates the function of another inflammatory
cell type so as to increase the uptake and rate of killing of I;_cruzi
by the latter, and imply that lactoferrin may play a role in host
defense against this parasite.

This research raises intriguing questions which deserve further
attention. For example,

What is the difference between amastigotes and trypo-

mastigotes that enables the former but not the latter to

express lactoferrin receptors and does this difference

assume biological significance with respect to parasite

survival and pathogenicity?

What are the precise signals elicited in the macrophage upon

binding of lactoferrin? Hhat enables these cells to display

greater cytotoxicity to I;_cruzi?

The answers to these questions will contribute to the further
understanding of the effects of lactoferrin in its modulation of

macrophage -.I; cruzi interaction, and also provide further insights

into the macrophage activation pathways in general.

   

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