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LSU Historical Dissertations and Theses
Graduate School
1996
Inflammatory Responses of the Jird to Brugia
Pahangi: Parasite Stage Specificity and Role of the
Macrophage.
Carmen Nasarre
Louisiana State University and Agricultural & Mechanical College
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Nasarre, Carmen, "Inflammatory Responses of the Jird to Brugia Pahangi: Parasite Stage Specificity and Role of the Macrophage."
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INFLAMMATORY RESPONSES OF THE JIRD TO
BRUGXA PAHANGI: PARASITE STAGE SPECIFICITY
AND ROLE OF THE MACROPHAGE
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Interdepartmental Program in Veterinary
Medical Sciences Through the Department
of Veterinary Microbiology
and Parasitology
by
Carmen Nasarre
D.V.M., University of Zaragoza, Spain, 1990
May, 1996
UMI Number: 9637798
UMI Microform 9637798
Copyright 1996, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized
copying under Title 17, United States Code.
UMI
300 North Zeeb Road
Ann Arbor, MI 48103
ACKNOWLEDGEMENTS
I am deeply indebted to my major professor, Dr. Thomas
R. Klei for his guidance and contribution to my efforts on
this dissertation.
His research creativity, great expertise
and knowledge in the field of parasite immunology have helped
greatly to the culmination of this work.
I would like to
thank Dr. Klei for the inspiration he has offered me both
personally and professionally,
as I strive to emulate his
manner and expertise.
Dr. James Krahenbuhl has also been instrumental in the
completion
of
this
work.
Without
his
support
and
the
assistance I gained from his mastery and knowledge in the
field of macrophage research this work would not have been so
readily accomplished.
Furthermore,
I would
like
to
extend
my
thanks
and
gratitude to the other members of my committee, Dr. Horohov,
Dr. Enright and Dr. Taylor for their constructive criticism,
wisdom, and invaluable advice.
Special
thanks go
to all
the members of Dr.
Klei's
laboratory, for their kindness, friendship and willingness to
help.
The mundane tasks of my research and experiments would
have not been completed without their time and effort.
I am very grateful to J.P. Pasqua for his time and help
in my experiments with Toxoplasma.
Julie Loesch and Linda
Adams have also been very helpful providing assistance and
advice.
ii
I gratefully acknowledge Cheryl Crowder and Mae Lopez
for
the
Michael
assistance
Kearney
in
for
the
the
histological
statistical
processing,
and
consultation
and
analysis.
I would like to express my most sincere gratitude to the
entire faculty in the department of Veterinary Pathology.
Each member has been
so kind
particular expertise with me.
to share
their wisdom
and
The sum of their combined
knowledge will stay with me always.
I am certain that I and
my work will both profit from their training and generosity.
Thanks are due to all my fellow graduate students, and
very especially to my good friend Galena Rybachuk with whom
I shared so much time of work and fun.
Finally, I wish to express my heartful gratitude to my
parents and sister for their continued love,
support throughout my education.
concern and
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .......................................... ii
LIST OF TABLES
............................................ v
LIST OF FIGURES ........................................... vi
ABSTRACT ................................................ viii
CHAPTER 1.
INTRODUCTION, LITERATURE REVIEW,
AND OBJECTIVES ................................ 1
CHAPTER 2.
DIFFERENTIAL INDUCTION AND REGULATION
OF INFLAMMATORY RESPONSES BY LIFE
CYCLE STAGES OF BRUGIA PA H A N G I ................ 37
CHAPTER 3.
EFFECT OF GAMMA RADIATION ON BRUGIA L3
DEVELOPMENT IN VIVO AND THE KINETICS OF
THE GRANULOMATOUS INFLAMMATION INDUCED
BY THESE PARASITES ............................69
CHAPTER 4.
MACROPHAGE ACTIVATION AND TNF PRODUCTION
IN JIRDS INFECTED WITH FEMALE OR MALE
BRUGIA P A H A N G I ................................ 90
CHAPTER 5 . CONCLUSIONS................................... 124
REFERENCES
VITA
.............................................. 129
153
LIST OF TABLES
Table 2.1
Worm recovery and microfilaria counts in
the peritoneal cavity of jirds inoculated
with B. pahangi L3 ....................... 48
Table 2.2
Parasite recovery from jirds inoculated
with male, female or L4 .................. 54
Table 2.3
Parasite recovery from jirds inoculated
with different numbers of adult worms ....62
Table 3.1
Parasite recovery from jirds inoculated IP
with normal or irradiated L3 at different
times post infection ..................... 76
Table 3.2
Lengths and development of B.pahangi
exposed to different doses of irradiation
or to no irradiation ..................... 78
Table 4.1
Effects of Con A MAF and murine rIFN-y on
the toxoplasmacidal activity of jird and
mouse macrophages ....................... 101
Table 4.2
Effects of BCG-PPD MAF on the
Toxoplasmacidal activity of jird and
mouse macrophages ....................... 109
Table 4.3
Nitric oxide production by jird and mouse
macrophages after in vitro treatment
with MAF, murine rIFN-y and/or LPS ......110
Table 4.4
Nitric oxide production by peritoneal
macrophages from BCG-PPD immunized jirds
and mice ................................. Ill
Table 4.5
Tumor necrosis factor-like production by
peritoneal macrophages from jirds infected
with female or male B.pahangi ........... 116
v
LIST OF FIGURES
Figure 2.1
Pulmonary granulomatous response to
Brugia pahangi antigen-coated beads in
jirds inoculated IP with L 3 ............... 44
Figure 2.2
Peritoneal macrophages in jirds inoculated
IP with L3 B. pahangi .................... 46
Figure 2.3
Peritoneal eosinophils in jirds inoculated
IP with L3 B. pahangi .................... 47
Figure 2.4
Pulmonary granulomatous response to
parasite antigen in jirds inoculated IP
with male, female or L4 B. pahangi ........50
Figure 2.5
Peritoneal macrophages in jirds inoculated
with male, female or L4 B.pahangi
....... 52
Figure 2.6
Peritoneal eosinophils in jirds inoculated
with male, female, of L4 B.pahangi ...... 53
Figure 2.7
Peritoneal microfilaria counts in jirds
inoculated with female worms or L4 .......56
Figure 2.8
Pulmonary granulomatous response to
parasite antigen in jirds inoculated IP
with dead or living worms
............... 57
Figure 2.9
Comparison of the pulmonary granulomatous
response to parasite antigen induced by
IP inoculation of high and low numbers of
female worms as well as MF ............... 59
Figure 2.10
Comparison of the pulmonary granulomatous
response in jirs inoculated with high or
low male worm numbers .................... 60
Figure 2.11
Peritoneal microfilariae counts in jirds
inoculated with different female worm
numbers or with MF ....................... 63
Figure 3.1
Pulmonary granulomatous response to
parasite antigen in jirds inoculated with
normal L3 or with L3 exposed to increasing
doses of gamma radiation ................. 79
Figure 3.2
Peritoneal macrophages in jirds inoculated
with normal L3 or with L3 exposed to
increasing doses of gamma r a d i a t i o n ......81
Figure 3.3
Photograph of a B.pahangi larva surrounded
by a peritoneal granuloma after 14 days
post infection ............................82
Figure 3.4
Photomicrograph of the granulomatous
inflammation induced by a B. pahangi
larva 14 days post infection ............. 84
Figure 4.1
Effect of BCG-PPD immunization on jird
macrophage activation measured by the
percentage of Toxoplasma infected
macrophages .............................. 102
Figure 4.2
Effect of BCG-PPD immunization on jird
macrophage activation measured by the mean
number of Toxoplasma per infected
macrophage ............................... 103
Figure 4.3
Effect of BCG-PPD immunization on jird
macrophage activation measured by the
mean number of Toxoplasma per 100
macrophages .............................. 104
Figure 4.4
Effect of BCG-PPD immunization on mouse
macrophage activation measured by the
percentage of Toxoplasma-infecteted
macrophage ............................... 105
Figure 4.5
Effect of BCG-PPD immunization on mouse
macrophage activation measured by the mean
number of Toxoplasma per infected
macrophage ............................... 106
Figure 4.6
Effect of BCG-PPD immunization on mouse
macrophage activation measured by the
mean number of Toxoplasma per 100
.............................107
macrophages
Figure 4.7
Macrophage activation in jirds inoculated
IP with female or male B.pahangi measured
by the percentage of Toxoplasma-infected
macrophages ..............................113
Figure 4.8
Macrophage activation in jirds inoculated
IP with female or male B.pahangi measured
by the mean number of Toxoplasma per
infected macrophage ..................... 114
Figure 4.9
Macrophage activation in jirds inoculated
IP with female or male B.pahangi measured
by the total number of Toxoplasma per 100
macrophages .............................. 115
vii
ABSTRACT
The host systemic and peritoneal inflammatory responses
against different stages of Brugia pahangi were compared in
intraperitoneally
responses
were
infected-jirds.
assessed
by
Systemic
measuring
inflammatory
the
pulmonary
granulomatous response to B. pahangi antigen-coated beads
(PGRN).
Peritoneal
inflammation
was
characterized
by
enumeration of different cell types in peritoneal exudates
following infection.
Further, the toxoplasmacidal activity
and TNF-a-like production of peritoneal macrophages were also
characterized.
Infection
with
L3,
L4,
male
worms,
female
worms,
microfilariae (MF) or dead adult worms induced a rapid PGRN.
This
response
decreased
to
levels
of
controls
in
jirds
inoculated with living parasites at the chronic time period,
indicating that viable worms are needed to downmodulate the
PGRN, but MF are not required.
Large numbers of female worms
induced greater downmodulation of the PGRN than low numbers,
and adult worm infection resulted in greater downmodulation
of the PGRN than MF inoculation.
These observations suggest
that parasite burden is important in filarial-induced PGRN
decrease.
The greatest peritoneal inflammatory response was
found in infections that resulted in MF production, and this
response did not correspond to the level of PGRN indicating
that
MF
act
as
potent
inflammatory
compartmentalization occurs.
viii
stimulus
when
Gamma radiation inhibited the development and decreased
the survival of B.pahangi L 3 .
This effect was inversely
related to the radiation dose used.
Downmodulation of the
PGRN occurred in infections with normal L3 or L3 irradiated
with 15 krads, but not in infections of L3 irradiated with
more than 15 krads, supporting the importance of the adult
stage in the PGRN downmodulation in absence of MF.
The PGRN
decrease occurred at the time period when the molt to adult
worms had just occurred, suggesting that larval stages are
also involved in the downregulation phenomenon.
Macrophages from jirds inoculated with male or female
B. pahangi
were
activated
to
kill
coinciding with the peak of PGRN.
56
DPI
and
decreased
markedly
Toxoplasma
at
15
DPI
TNF production peaked at
at
135
DPI.
Absence
of
toxoplasmacidal activity was found in macrophages after 56
DPI corresponding to PGRN decrease.
These data suggest that
filaria-specific hyporesponsiveness may be associated with a
downmodulation of macrophage function.
CHAPTER 1.
INTRODUCTION, LITERATURE REVIEW AND
OBJECTIVES
Human lymphatic filariasis is a long-term,
disease
principally
caused
Wuchereria bancrofti
by
the
filariid
and Brugia malayi.
Brugia
parasitic
nematodes
timori
responsible for an additional small percentage of cases.
is
The
estimated prevalence of lymphatic filariasis is currently 120
million
(WHO,
Wuchereria
Pacific,
1992;
bancrofti
Ottesen
exists
as well as
and
in
Ramachandran,
Asia,
Africa,
1995).
the
South
in coastal areas of Brazil and some
Caribbean islands, such as Haiti.
Brugia malayi occurs in
Malaysia, the Philippines, and some foci in India.
Brugia
timori is limited to the Indonesian islands of Timor and
Flores (Denham and McGreevy, 1977; Ottesen, 1984a).
Brugia pahangi
is a closely related species that
found in domestic and wild carnivores in Southeast Asia.
is
B.
pahangi has been reported as a natural infection in a few
people from South Kalimantan,
1985).
Indonesia
(Palmieri et al.,
However, the significance of this filarial species as
a human parasite is still undetermined, since the diagnosis
of
these
human
characteristics
cases
of
activity and was not
the
was
based
microfilarial
on
the
acid
staining
phosphatase
confirmed by detailed morphological
features of adult parasites or by successful development of
microfilariae
to infective
larvae
1
in the mosquito.
B.
2
pahangi has been experimentally transmitted to humans (Edeson
et al., 1960).
PARASITE LIFE CYCLE
In the definitive host,
lymphatic vessels
legs,
arms,
of
adult nematodes
testis,
breasts,
epididymis,
and
live in the
spermatic
cord,
pelvic
and
abdominal,
retroperitoneal regions as well as in associated regional
lymph nodes.
(MF)
that
The mature female worms produce microfilariae
migrate
to peripheral
blood.
When
ingested by
suitable mosquito intermediate hosts, MF develop to infective
third stage larvae (L3).
mouthparts
and enter
produced by the vector.
The L3 migrate from the mosquito
the definitive
host
thru
the wound
Once in the skin, the L3 migrate to
the lymphatics and lymph nodes to molt to the fourth stage
larvae (L4) which develops and finally molts again to become
mature female and male worms (Taylor, 1960; Schacher, 1962b).
The prepatent period is approximately 3 to 8 months depending
on
the
parasite
species.
Thus,
L3,
L4,
adults
and
MF
interact with the host immune system at different times and
locations in the body.
Furthermore,
each of these larval
stages is biochemically, morphologically and antigenically
diverse and each exists for different durations during the
life cycle (Ottesen, 1979; Partono, 1987).
Therefore, stage-
specific responses may occur during filarial infection and
may
be
patterns
in
of
part
the
responsible
disease
for
(Maizels
the
and
different
Lawrence,
clinical
1991).
3
Adaptation
of
transmission
filarial
has
parasites
probably
lead
to
to
accomplish
the
optimal
development
strategies for prolonged survival in the host.
of
For instance,
adult worms may survive up to 30 years in humans, releasing
MF throughout
1995).
this
time into the blood stream
(Nutman,
The persistence of filarial parasites in their mammal
hosts constitute an important hallmark of filarial infection.
CLXNXCOPATHOLOQICAL MANIFESTATIONS
Lymphatic filariasis is a spectral disease;
infection
results in different clinical outcomes or conditions, each of
which has been correlated with diverse states of filarial
specific immune
Nutman, 1991).
responsiveness
(Ottesen,
1984b;
King and
In endemic regions, infected individuals can
be classified in five different categories (Ottesen, 1980).
1.
Microfilaremic asymptomatic patients have no apparent
clinical manifestations, have circulating MF in peripheral
blood and are considered immunologically hyporesponsive to
the parasite.
These individuals normally constitute
the
largest group of the infected population and represent an
important
reservoir
Recently,
the
detected
use
for
of
morphologic
transmission
lymphatic
changes
of
imaging
such
as
the
parasite.
techniques
dilatation
has
and/or
tortuosity of the lymphatic vessels (Case et al., 1992; Witte
et al., 1993) in some of these microfilaremic asymptomatic
patients.
Renal
(Dreyer, 1992).
abnormalities
have
also
been
reported
4
2. Acute filariasis patients are those patients with
periodic onsets of acute lymphatic obstruction manifested as
lymphangitis,
lymphadenitis
and
transient
lymphedema
accompanied by systemic symptoms of fever and malaise.
These
acute episodes, also known as "filarial fevers", last for a
few days and may recur at irregular intervals of as much as
1 year
duration.
microfilaremic.
These
may
or
may
not
be
When these onsets repeat with increasing
frequency patients
chronic disease.
individuals
may develop persistent
lymphedema
and
Unfortunately, this group of patients has
not been studied in detail.
3. Chronic lymphatic pathology patients are those that
have developed chronic lymphatic obstruction manifested as
elephantiasis, hydrocele or/and chyluria.
Hydrocele is the
accumulation of lymph between the tunicas that surround the
testes.
Chyluria is the excretion of chyle in the urine.
Generally, these patients show pronounced filarial specific
immune responses and amicrofilaremia.
However,
can
and
be
clinically,
heterogeneous.
parasitologically
this group
immunologically
For instance, hydrocele patients are often
microfilaremic when compared to elephantiasis
individuals
that rarely are (Lammie et al., 1993; Addis et al., 1995).
4. Tropical pulmonary eosinophilia occurs in a small
percentage
of
filarial
characterized by allergic,
infected
individuals
and
is
asthma-like pulmonary symptoms,
apparently unrelated to lymphatic pathology.
These patients
5
are hypersensitive to filarial antigens, particularly those
derived from MF and show
of
serum
IgE
and
diethylcarbamazine
Inadequately
or
amicrofilaremia.
effectively
non-treated
interstitial fibrosis.
obscure,
allergic
this
elevated eosinophilia, high levels
syndrome
sensitization
Treatment
resolves
cases
with
the
symptoms.
to
pulmonary,
evolve
Although the pathogenesis is still
could
to
be
MF
explained
antigens,
as
a
severe
resulting
in
entrapment and clearance of MF in the lungs and other organs
of the reticuloendothelial system such as spleen and lymph
nodes (Ottesen and Nutman, 1992) .
5.
Endemic normals are individuals who are continuously
exposed to infected mosquitoes, have circulating filarialspecific antibodies, but have no clinical signs or evidence
of infection and are considered putatively immune.
group,
however,
is difficult to define.
This
Strict clinical,
parasitological and serological criteria must be applied in
an
endemic
population
those
to
distinguish
that
occult
or
resistant
individuals
from
infections.
Some detailed studies have reported the endemic
normal as a rare individual
harbor
truly
(Freedman et al.,
low
1989;
grade
Day,
1991) . Furthermore, it has been hypothesized that protective
resistance
may
result
primarily
from
a
dynamic
process
associated to continuous reinfection rather than to naturally
acquired immunity (Day, 1991) .
Although most of the patients fall into one of these
categories, individuals with overlapping syndromes do exist.
Chronic lymphatic pathology patients with microfilaremia have
been
identified.
In
these
patients
immunoregulatory
mechanisms that operate are more difficult to explain (Lammie
et al.,
1993).
Studies by Lammie
et al.
(1993)
in the
Haitian community of Leogane have revealed that many of the
patients
with
hydrocele
were
microfilaremic
(25
of
42) ,
meanwhile only 1.5% of patients with chronic limb involvement
had MF in peripheral blood.
Microfilaremic individuals with
hydrocele lacked antifilarial immune reactivity suggesting
that pathogenesis of hydrocele formation may be initiated by
mechanisms other than immune or inflammatory reactions.
Data
(Lammie et
that
al.,
1993;
Addis
et al.,
1995)
microfilaremic patients with hydrocele are
indicate
in an earlier
stage of the disease than amicrofilaremic hydrocele patients
supporting the hypothesis that microfilaremic asymptomatic
individuals progress to an amicrofilaremic immune responsive
state
(Bundy et al., 1991).
Interestingly,
clinical signs of lymphatic filariasis
appear more rapidly and are more severe when the infection
occurs in individuals that have not been exposed previously
to the parasite.
native
to
documented
This occurs in individuals that are not
endemic
when
areas.
thousands
This
of
phenomenon
American
was
first
soldiers
moved
temporarily to the South Pacific area during World War II.
7
Approximately one third of soldiers exposed to filariasis
developed acute
onsets of
lymphangitis,
lymphadenitis
or
genital inflammation with absence of microfilaremia (Wartman,
1947; Ottesen, 1980; Partono, 1987).
affected soldiers
Symptoms remitted when
left endemic areas.
Similar responses
occurred in Indonesians of the same ethnic background who
moved
from
filariasis
filariasis
(Partono
free
et al.,
areas
to
1978).
areas
When
endemic
these
for
affected
individuals leave filariasis prevalent areas the clinical
signs tend to resolve.
However, if they remain continuously
exposed to infective larvae, they develop a more rapid and
severe form of elephantiasis (Wartman, 1947; Partono et al.,
1978).
Exposure to parasites when the immune system is not
fully competent as may occur during pre- or neo-natal life
may
result
in
specific
filarial
immune
tolerance
with
subsequent attenuation of disease manifestations (Nutman et
al.,
1986; Ottesen,
1989).
Thus,
differences in response
among individuals of similar ethnic background can be partly
explained in this manner.
The clinical manifestations of Bancroftian filariasis
differ in some aspects with those of Brugian filariasis.
In
Bancroftian filariasis clinicopathologic changes are more
extensive affecting the whole leg and arm, and also commonly
occur in the male genitalia.
involvement
of
male
During the acute period the
genitalia
lymphatics
leads
to
funiculitis, epididymitis and orchitis, manifested clinically
8
by pain, swelling and tenderness.
of
orchitis
Hydrocele
and
is
epididymitis
the
Repeated, acute episodes
often
commonest
result
chronic
in
hydrocele.
manifestation
of
Bancroftian filariasis in many parts of the world (Partono,
1987).
Microfilariae are sometimes present in the hydrocele
fluid, even though they may be absent from peripheral blood.
Some
infected
individuals
develop
chyluria,
which
is
associated with blockage of the retroperitoneal lymph nodes
below the cisterna chyli resulting in efflux and flow of the
mesenteric lymphatics
directly into the renal lymphatics.
The urine of patients with chyluria contains large amounts of
dietary lipids, proteins and possibly fat-soluble vitamins,
leading
to weight
persistent,
loss and debilitation.
If severe and
chyluria may also result in lymphopenia due to
loss of lymphocytes in the urine (WHO, 1985).
In Brugian filariasis elephantiasis of the legs occurs
below the knee and only occasionally affects the arms below
the elbow.
Genital
involvement rarely occurs
(Review in
Denham and McGreevy, 1977).
PATHOGENESIS AND PATHOLOGY OF LYMPHATIC FILARIASIS
Experimental
studies
in
laboratory
animals
and
observations in natural human infections have helped in the
understanding of
lymphatic
lesion pathogenesis
caused by
filarial parasites
(Ewert et al., 1972; Gooneratne, 1973).
After
the
penetrating
skin,
the
L3
locate
in
afferent
lymphatic vessels, and subcapsular and cortical sinuses of
9
lymph nodes where they molt twice.
The presence of
the
parasites, their cast-off sheaths, and their molting fluids
and other secretory/excretory products presumably initiate
morphological changes in the vessel characterized by luminal
dilatation or 11lymphangiectasia", endothelial
proliferation
and thickening of the lymphatic wall with inflammatory cells
and fibrosis
(O'Connor,
1932).
Adult parasites probably
cause an important mechanical damage to the lymphatic vessel
wall and valves especially due to their continuous movement
leading to lymph vessel dilatation (Von Lichtenberg, 1987).
More
severe
lymphatic
granulomatous
reactions
occlusion
the
of
damage
occurs
resulting
vascular
when
worms
in partial
lumina.
or
Granulomas
induce
complete
are
most
typically associated to the presence of dead or dying adult
worms.
However, MF and other parasite products are probably
involved in this inflammatory process as well (Meyers et a l .,
1976; Klei et al., 1982; Von Lichtenberg, 1987).
often
surround
parasites
and
are
Granulomas
characterized
by
accumulation of macrophages, eosinophils, lymphocytes, plasma
cells,
and multinucleated giant cells with fibrous tissue
proliferation developing in the periphery.
Other times, more
exudative reactions occur and consist of areas of necrosis
with polymorphonuclear leukocytes, especially eosinophil, but
also neutrophil infiltration surrounding the worms or worm
remnants (O'Connor and Hulse, 1935; Von Lichtenberg, 1987).
Lymphatic vessel dilatation with no inflammation may be the
only
histopathologic
change
accompanying
the
worms.
Sometimes a mild non-granulomatous, parietal infiltrate may
be present in the lymphatic vessel walls
1991).
(Jungmann et al.,
These slight changes may reflect a suppression of
filarial specific immune reactivity.
The damage of valves,
along with the lymphatic dilatation, lymph stasis and lymph
vessel
thrombosis
by
granulomas
result
in
increased
hydrostatic pressure and increased permeability of the walls
to the lymph.
Consequently, there is leakage of fluid with
a high concentration of protein into the adjacent tissues.
Protein-rich lymph in the tissues is a potent stimulator of
fibrogenesis and neovascularization.
These latter processes
are also favored by the inflammatory reaction that the worms
and their products induce.
The mechanism of fibrosis in
filariasis is not well understood, but is probably associated
with both mechanical blockade of lymph flow and parasiteinduced inflammation.
in
rabbits
Hammerberg,
Surgical ligation of lymphatic ducts
(Wolfe
et
al.,
1989)
induced
1983)
mild
and
dogs
lymphedema
(Snowden
with
and
little
fibrotic changes indicating that fibrosis in elephantiasis
patients is a more complex process.
Several
authors
have
proposed
that
hypersensitivity
reactions mounted against parasites and their products are
important factors leading to severe inflammation (Rifkin and
Thompson, 1945; Lichtenberg, 1957).
Thrombolymphangitis and
thrombophlebitis with parasite material diffusing into the
11
interstitium
and exacerbating the inflammatory process have
been interpreted as a phenomenon generically related to the
Arthus
reaction
circulating
(Lichtenberg,
immune
1957).
complexes
and
Further,
enhanced
increased
complement
consumption have been found in individuals with elephantiasis
when
compared
with
asymptomatic
(Gajanana et al., 1982;
microfilaremic
patients
Prasad et al., 1983; Nath et al.,
1991).
Tissues affected with lymphedema are likely to be more
prone to bacterial infections,
since the normal texture of
the skin covering affected locations may be altered and the
underlying
compromised
lymph
remove incoming pathogens.
drainage
cannot
effectively
Bacterial complications lead to
more severe pathology and have been reported to form part of
many filarial cases
(Olszewski et al.,
1990;
G.Dreyer in
Maizels et al., 1995).
Finally, it should be pointed out that the scarcity of
autopsies in human filariasis patients has greatly hampered
the understanding of lesion development in this disease.
IMMUNE RESPONSES IN HUMAN LYMPHATIC FILARIASIS
The different clinical outcomes of filarial infection
are presumably related to the capacity of the host immune
system to respond to filarial antigens (Ottesen, 1980; King
and Nutman, 1991).
have
Asymptomatic microfilaremic individuals
impaired immune responses
to filarial antigens when
compared with the strong immune responsiveness of individuals
12
with lymphatic pathology or tropical pulmonary eosinophilia,
who are usually amicrofilaremic (Ottesen et al., 1977).
Low
lymphocyte blastogenic responses to filarial antigen have
been
consistently
found
in
microfilaremic
asymptomatic
individuals in contrast with the remarkably high lymphocyte
proliferative responses of individuals showing pathology and
amicrofilaremia (Ottesen et al., 1977; Piessens et al., 1980;
Nutman et al.,
antibody
1987a;
levels
King et al.,
also
differ
1992).
between
Antifilarial
microfilaremic
and
amicrofilaremic chronic filariasis patients. Hyporesponsive
microfilaremic asymptomatic individuals have low levels of
IgGl, IgG2 and IgG3
1982)
(Nutman et al., 1987b; Ottesen et al.,
but have high levels of IgG4
Hussain et a l ., 1987).
1985;
Conversely, amicrofilaremic pathology
patients produce high levels of
little or no IgG4
(Ottesen et al.,
IgGl,
(Hussain et al.,
IgG2
and
IgG3
and
1987; Maizels et al.,
1995) . In microf ilaremic asymptomatic patients, antifilarial
IgG4 response correlates with microfilaria counts (Kwan-Lim
et al., 1990) and levels of this isotype drop markedly after
treatment with the microfilaricidal drug diethylcarbamazine
(Maizels et al.,
1995;
Sartono et al.,
1995).
The
IgG4
subclass has been demonstrated to possess blocking activities
that would prevent IgE-mediated hypersensitivity reactions
(Aalberse et al., 1983).
As
observed
may
in
be
expected,
cytokine
important
profiles
differences
between
defined
are
also
patient
13
populations.
Peripheral
lymphocytes
of
microfilaremic
asymptomatic patients produce high levels of IL-4 and IL-S in
response to filarial antigens but make low levels of IFN-y,
IL-2 and GM-CSF.
In contrast,
lymphocytes of symptomatic
amicrofilaremic individuals respond with increased levels of
IFN-y and IL-2 production (Nutman et al., 1987a; King et al.,
1993).
IL-10
asymptomatic
pathology
has
been
observed
microfilaremic
patients,
and
to
patients
most
be
elevated
compared
importantly,
to
in
chronic
neutralizing
antibodies against IL-10 and TGF-beta restore the in vitro
hypo-responsiveness
to
parasite
antigens
asymptomatic microfilaremic patients,
However,
present
(King et al.,
in
1993).
others investigators have been unable to restore
unresponsiveness of cultured T cells by adding anti-IL-10
(Maizels et al., 1995).
(DEC)
and
ivermectin
Treatment with diethylcarbamazine
restores
immune
responsiveness
in
asymptomatic microfilaremic patients (Piessens et al., 1981;
Lammie et al.,
1992).
patients
treated
are
responsiveness
infections,
is
Interestingly,
with
enhanced
DEC
after elephantiasis
anti-filarial
suggesting
that
all
immune
filarial
not necessarily including microfilaremia,
are
able to induce some degree of immunodepression (Maizels et
al.,
1995).
Other
studies
have
also
found
that
some
asymptomatic amicrofilaremics and some elephantiasis patients
manifest specific hyporesponsiveness presumably due to active
infection (Yazdanbakhsh et al., 1993).
King et al.
(1993)
14
believe that modulatory cytokines may be responsible for the
up or down regulation of the immune responses in filarial
infected individuals, by responding either with Thl or Th2
cytokine profiles,
respectively.
Based on this hypothesis,
pathology would manifest when Thl
cytokines predominate,
whereas the appearance of a Th2 phenotype would lead to a
hyporesponsive state typical of microfilaremic asymptomatic
individuals.
Conversely, Maizels et al.
(1995) argue that
high T-cell responsiveness, release of Thl cytokines and IgE
production
may
not
elephantiasis.
be
implicated
in
authors
have
These
elephantiasis
patients
exhibit
the
a
development
found
that
of
some
microfilaremic-like
phenotype, with high IgG4 levels and hyporesponsive T cells.
Furthermore,
reduces
long-term
symptoms
treatment
(Partono
et
al.,
with
diethylcarbamazine
1984)
and
results
in
restoration of T cell hyperresponsiveness and release of
cytokines,
immune
such as
factors
IFN-y,
contradicting again that
lead to pathology
Sartono et al., 1995).
(Maizels
et
al.,
these
1995;
Moreover, many endemic normals have
levels of IgE and T-cell responses similar to elephantiasis
patients without showing any clinical signs.
However, it may
be important to note that levels of IgGl, IgG2 and IgG3 in
endemic normals are always lower than in chronic pathology
patients
(Maizels
especially
IgG3,
participate
in
et
al.,
are
good
immune
1995).
at
complex
fixing
These
IgG
complement
reactions.
isotypes,
and
Deposition
may
of
15
immune
complexes
in
filariasis
has
not
been
closely
investigated, but it has been proposed by some authors as a
relevant factor in pathology (Lichtenberg, 1957; Klei et al.,
1982; Hussain et al., 1987; Nath et al., 1991).
It has been suggested that endemic normals are able to
kill incoming L3 preventing establishment of worms in the
lymphatic system and initiation of pathology
Lawrence, 1991).
(Maizels and
One immunological study in an endemic area
in the Pacific revealed that filarial exposed non-infected
children are more responsive than the equivalent group of
adults, suggesting that the so called "endemic normals" may
become
specifically desensitized with continuous exposure
eventually
being
hyporesponsive
able
state
to progress
(Ottesen et
to
al.,
the
microfilaremic
1977).
Long-term
studies in this filariasis group would help enormously in the
understanding
of
the
immune
response
dynamics
and
risk
factors for development of pathology or immune tolerance.
Pre- or peri-natal exposure to filarial antigens has
been proposed to be partly responsible
immune tolerance in filariasis
for the
state of
(Weil et al., 1983).
Anti-
filarial IgM (Dissanayake and de Silva, 1980) and IgE (Weil
et al.,
1983)
have been found in the cord blood of human
fetuses and microfilariae have occasionally been found in the
bloodstream of newborns
(Loke,
1982).
Moreover,
children
born from microfilaremic mothers are more prone to become
microfilaremic
than
children
born
from
amicrofilaremic
16
mothers
(Lammie et al., 1991).
Studies in Brugia-infected
jirds have demonstrated that offspring born from infected
mothers have higher levels of microfilaremia and develop less
severe pathology than the progeny from uninfected jirds (Klei
et a l ., 1986).
Monocytes
factors
(Piessens
(Piessens
lymphocytes
possible
al.,
al.,
or
1982)
suppressors
patients.
1980),
1980)
(Piessens et al.,
active
filariasis
et
et
However,
in
serum suppressor
suppressor
CD8+
T
have been reported as
studies
other
with
similar
Brugian
studies
in
Bancroftian filariasis patients have failed to demonstrate
such suppressor cell populations (Nutman, et al. 1987a).
Few studies have addressed host genetic influences on
the
immune
Ottesen
et
responses
al.
and
(1981)
outcomes
observed
of
filarial
familial
disease.
clustering
of
infection in studies performed in Polynesia but failed to
find any linkage
to HLA-A or B-locus
specificities.
An
association between chronic lymphatic pathology and HLA-B15
has been found in patients from areas of South India and Sri
Lanka where filariasis is endemic (Chan et al., 1984) . More
recently, M. Yazdanbakhsh et al.
have
found
that
one
class
II
progression to elephantiasis.
(in Maizels et al.,
allele
is
1995)
correlated with
In addition,
these studies
have revealed that asymptomatic microfilaremic individuals
have higher frequency of 2B3 (epitope present on DQW6, 8 and
9) than lymphatic pathology patients (Maizels et al., 1995).
17
EXPERIMENTAL ANIMAL MODELS
Advances in our understanding of filarial pathogens is
highly dependent on animal models, especially because of the
parasite
longevity,
the difficulty to determine
parasite
burdens in vivo, the inavalaiblity of autopsy specimens and
the impossibility of performing longitudinal studies, since
treatment
to
effective.
clear
infection
Therefore,
must be treated,
evolution of
is
readily
available
and
patients diagnosed with filariasis
making it impossible to investigate the
the diverse
clinical
conditions.
The
main
constraint in filariasis research is the lack of an adequate
experimental model which is susceptible to infection and at
the same time is well defined immunologically..
known
animal
in
which
Wuchereria
bancrofti
patency is the silvered leaf monkey,
(Palmieri et al., 1980).
The only
develops
Presbytia
to
cristatus
The difficult availability of this
monkey species makes its use as an animal model not possible.
A
variety
pahangi.
dog
of
animals
are
permissive
for
B.
malayi
and
These include, the cat (Denham and Fletcher, 1987),
(Schacher and Sahyoun,
1967), mongolian jird
(Ash and
Riley, 1970a), immunodeficient mice (Suswillo et al., 1980)
and ferret (Crandall et al., 1982), which have been used for
the study of filariasis.
rat strains
Other animal models, such as some
(Cruickshank et al., 1983), the golden hamster
(Ash and Riley, 1970b; Malone et al., 1974), the guinea pig
(Ahmed,
1967),
and
the
rabbit
(Ahmed,
1967)
have
less
18
consistent
permissiveness
and
excessive
susceptibility to Brugia infection.
been
limited.
Immunocompetent
variation
in
Therefore, their use has
mice
do
not
support
the
development of Brugia spp. to patency (Chong and Wong, 1967;
Howells et al., 1983).
However, they allow development of
early larval stages before reaching maturity and short-term
survival of inoculated microfilariae
The
inoculation
of
nonpermissive model,
different
(Grove et al., 1979).
life
cycle
stages
in
a
although informative, may not totally
represent the real situation.
The jird is probably a more
suitable model to investigate the role that each of the life
cycle stages plays in the induction and downregulation of
immune and inflammatory responses during filarial disease.
In this model,
the time periods at which the life cycle
stages molt and the adults release MF have been accurately
defined.
Furthermore,
studies
have
demonstrated
that
intraperitoneal and subcutaneous infections result in similar
lesions (Jeffers et al., 1987).
Cat
Cats and dogs are natural hosts for B. pahangi and B.
malayi in areas of Southeast Asia.
Filarial infection in
cats closely resembles that caused by Wuchereria and Brugia
in humans, and provides important clues for the understanding
of the disease dynamics and immunity (Grenfell et al., 1991) .
In
primary
establish
infections
successfully
the
in
adult
the
worms
lymphatic
that
survive
system
are
and
long
19
lived, and microfilaremias remain stable in most cats (Denham
and Fletcher,
first
1987).
suggested
Although,
the
patterns of worm recovery
development
against incoming L3
of
concomitant
(Grenfell et al.,
1991),
immunity
more recent
parasitological observations contradict this interpretation.
Interestingly, amicrofilaremic cats appear to be able to also
kill
adult
parasites
(Denham
et
al.,
1993).
A
small
proportion (up to 15%) of cats with primary infections and a
larger
proportion
infections
clear
Fletcher, 1986).
manifest
Rogers,
(up
a
their
animal model
40%)
of
cats
microfilaremias
with
repeated
(Denham,
1983;
Those cats with repeated infections often
transient,
1975).
to
The
recurrent
clearance
lymphedema
(Denham
of microfilaremia
in
and
this
coincides with the peak production of anti-
microfilarial antibodies (Ponnudurai et al., 1974) mimicking
a similar response in human patients (Grenfell et al., 1991;
Maizels
and Lawrence,
1991).
In conclusion,
the
cat-B.
pahangi model supports the hypothesis that the development of
pathology is related to the amicrofilaremic state and the
appearance of immunity.
similarity
chronic
in disease
lymphatic
However,
dynamics,
pathology
in spite of the apparent
cats
seen
do not
in
develop
human
the
lymphatic
filariasis (Denham et al., 1994).
Dog
The dog is a natural host for B. pahangi and a good
model to study lymphatic filariasis.
Infected dogs develop
20
a
variety
of
filariasis
clinical
spectrum
changes
(Snowden
that
parallel
the
and
Hammerberg,
human
1989).
Pathologic changes in the dog appear to be more severe and of
earlier manifestation than in the cat (Schacher and Sahyoun,
1967; Schacher et al., 1969).
Furthermore, transient limb
lymphedema and lymphadenopathy is not necessarily associated
with multiple infections (Snowden and Hammerberg, 1989) .
Ferret
Ferrets are particularly hyperresponsive to infection
and develop a syndrome more characteristic of hyperresponsive
patients manifesting TPE or filarial fevers,
or those non
endemic populations that are primarily exposed (Hines et a l .,
1989).
Subcutaneous
infection of ferrets with B. malayi
results
in establishment of adult worms
in the lymphatic
system and production of microfilariae after approximately
three months.
Most ferrets become amicrofilaremic before
eight months postinfection and only a small percentage (10 to
15%)
develop
prolonged
microfilaremia.
Clearance
microfilaremia is associated with eosinophilia,
immunoresponsiveness
to
multifocal granulomas.
liver,
lung,
inflammatory
filarial
antigens
and
of
prominent
widespread
These granulomas occur principally in
and lymph nodes and are characterized by an
infiltrate
of
primarily
macrophages
and
eosinophils with occasional entrapped dead MF and a typical
Splendore-Hoeppli material that closely resembles the MeyersKourewnaar
bodies
described
in
TPE
lesions
of
humans
21
(Crandall et al., 1982; Crandall et al., 1984; Hines et al.,
1989).
Following a single infection lymphedema of the legs
is only occasionally apparent
ferrets
receive
multiple
and always transient. When
infections,
those
that
become
amicrofilaremic often develop sustained lymphedema of the
legs and do not become patent on reinfection
al., 1987).
animals
Lymphedema has been observed to persist in some
for
more
microfilariae
infection
(Crandall et
and
than
induce
three
partial
years.
Inoculations
resistance
promotes development
of
to
of
challenge
lymphatic
disease
coinciding with the clearance of microfilariae (Crandall et
al., 1990).
to
be
Thus, acquired resistance in the ferret appears
associated
with
an
amicrofilaremic
state.
The
lymphangitis and lymphatic dilatation that occurs in ferrets
is similar to that observed in other experimental hosts and
humans (Hines et al., 1989).
in
studies
resistance
implication
to
clarify the
and
development
of
The ferret model may be useful
relationship
of
certain parasite
between
pathology as
stages,
well
such
as
acquired
as
MF,
the
in
disease and immunity.
Mouse
The murine model is particularly advantageous to study
specific
cellular
and
humoral
of
the
responses
availability
during
of
filarial
infection
because
immunological
reagents.
Although immunologically intact mice are not fully
permissive for Brugia spp. infection; depending on the route
22
of inoculation and the life cycle stages used, parasites may
survive long enough to be studied immunologically (Lok and
Abraham,
1992).
In contrast,
infective larvae develop to
full maturity in athymic nude mice
and
in
scid
mice
(Nelson
et
(Suswillo et al., 1980)
al.,
1991).
However,
reconstitution of immunodeficient mice with T cells confers
the
capacity
to
mount
effective
humoral
and
cellular
responses to the parasite (Vickery et al., 1983; Vickery and
Vincent, 1984) which indicates that T cells are important in
resistance.
The
immunodeficient
murine
model
has
also
provided important insights regarding lesion pathogenesis.
The
use
of
existence
the
of
immunodeficient
mice
two possible mechanisms
has
revealed
of disease;
the
one
is
independent of the immune response and the result of the
presence
of
the
immunodeficient
parasites
mice,
where
in
the
they induce
lymphatics
endothelial
of
cell
proliferation, severe lymphatic vessel dilatation and finally
lymphedema and elephantiasis (Vincent et al., 1984; Nelson et
al., 1991).
The second is dependent on the immune response
and occurs when immunodeficient mice are reconstituted with
cells
from
antigens
around
resulting
in
the parasites
obstruction,
1991).
immunocompetent mice
sensitized with
pronounced
that
leads
inflammatory
to
lymphatic
lymphedema and elephantiasis
filarial
responses
damage
and
(Vickery et al.,
Studies in mice have contributed information on the
types of immune responses mounted against filarial parasites.
23
Several studies
1993a;
(Bancroft et al.,
Lawrence
et
al.,
1993;
1994)
have
Pearlman et al.,
shown
that
these
immunocompetent animals develop local and systemic dominant
Th2 responses after inoculation of Brugia MF, MF extracts,
irradiated L3 or adult worms.
Acquired resistance to MF
inoculated intravenously or intraperitoneally occurs.after
subcutaneous sensitization with MF antigens and correlates
with a Th2 response (Pearlman et al., 1993b).
L3
challenge
irradiated
response
L3,
is
accomplished
and
(Hayashi
this
et
after
again
al.,
is
1989;
Resistance to
vaccination
associated with
Bancroft
et
al.,
with
a Th2
1993).
Intraperitoneal inoculation of adult female or male worms
induce
a
rapid
Th2
response,
whereas
results in an early Thl phenotype
Interestingly,
studies
in
IL-4
inoculation
of
MF
(Lawrence et al., 1994).
deficient
mice
inoculated
intraperitoneally with different stages of B. malayi indicate
that the survival of adult worms, MF or L3 appears not to be
affected by a Th2 response (Lawrence et al., 1995) . The role
of IL-13, however, has not been investigated.
Jird
Jirds support complete development of both B. malayi and
B. pahangi
(Ash and Riley,
1970a; Ash and Riley,
1970b) .
However, B. pahangi has been used more extensively than B.
malayi for experimental infections in the jird because the
prepatent period is shorter, the microfilaremia is of higher
levels and the location of adult worms resembles more closely
24
that reported for natural hosts (Ash, 1973).
Furthermore, no
significant pathological or immunological differences were
observed in a comparative study of jirds infected with B.
malayi
or
B.
pahangi
(McVay
et
al.,
1990).
In
jirds,
subcutaneous inoculation of L3 in the inguinal area results
in localization of the parasites in lumbar, spermatic cord
and
testicular
lymphatics,
and
associated
lymph
nodes.
Pathological findings resemble those reported in humans and
other experimental animals
(Ah and Thompsom, 1973; Vincent
et al., 1980; Connor et al., 1986; Klei et al., 1986).
The
pathological changes in the jird lymphatics are characterized
by
lymphatic vessel
dilatation,
inflammatory and
fibrous
thickening of the vascular wall and typical intralymphatic
granulomatous
lymphedema
or
thrombi
(Ah and Thompsom,
elephantiasis,
as
seen
1973).
However,
in human
filariasis, are not observed in the jird.
lymphatic
First evidence of
lymphatic inflammation appears to coincide with the final
molt of worms to become adults (Vincent et al., 1980) .
The
kinetics of the inflammatory response in the lymphatic system
and the granulomatous reactivity around Brugia antigen-coated
beads embolized to the lungs have demonstrated the presence
of several states in the jird-B. pahangi model that mimic
some of the conditions discussed in human filariasis.
include,
the
hyporesponsive
antigen
acute
responsive
state,
immunization,
state,
a hyperresponsive
and
an
immune
the
These
microfilaremic
state
state
induced by
induced
by
25
vaccination (Klei et al., 1981; Weil et al., 1983; Yates and
Higashi,
1985; Kazura et al., 1986).
Numbers of lymphatic
granulomatous lesions are greatest between 60 and 90 days
postinfection
(Vincent
significantly thereafter
1990).
The
et
al.,
1980),
(Klei et al.,
decreasing
1988; Klei et al.,
antigen reactivity in the lungs measured by
granuloma areas around antigen-coated beads (PGRN) is maximum
at 14 DPI
(Rao et al.,
1995) .
This period of peak PGRN
coincides with the maturation of larval
early adult worms.
stages to become
PGRN decreases markedly as the infection
progresses to the chronic period (Klei et al., 1981).
downregulation
occurs
before
adult
worm
This
maturation
and
patency, and is associated with the presence of mature adult
worms within lymphatics and with the onset and persistence of
MF.
Blastogenic responses of renal lymph node cells peak
early
1995).
in
infection and decrease
thereafter
(Rao et
a l .,
Kinetics of axillary lymph node cell proliferation is
strikingly different
from that of renal
lymph node cells
draining the site of infection in that no downmodulation is
observed in the former.
Proliferative responses of spleen
cells peak later than those of renal lymph node cells, and
decrease
similarly
in
the
chronic
state
of
infection
coinciding with persistent microfilaremia (Lammie and Katz,
1983;
Leiva and Lammie,
decreased
splenic
1989;
Klei et al.,
responsiveness
has
been
1990).
shown
This
to
be
associated with the presence of an adherent cell population
26
in the spleen
antibody
(Lammie and Katz,
levels
increase
1984).
gradually
Filarial-specific
during
infection
remain elevated throughout the chronic state
1990).
and
(Klei et al.,
Peripheral blood eosinophils peak during the acute
period coinciding with the molt to the adult stages,
decrease significantly during the chronic period
al., 1990; McVay et al., 1990) .
and
(Klei et
Converse to what is seen in
ferrets and cats, multiple challenge infections do not induce
protection in microfilaremic jirds, and do not significantly
modify preexisting lymphatic lesions or reactivity around
antigen-coated
Likewise,
beads
in
the
lungs
chronically infected,
(Klei
et
al.,
amicrofilaremic
1988).
jirds are
susceptible to reinfection and challenge infection does not
increase lymphatic pathology (Lin et al., 1995).
resistance
Protective
can be achieved in jirds by vaccination with
irradiated L3
(Yates and Higashi, 1985; Chusattayanond and
Denham, 1986; Petit et al., 1993).
Interestingly, vaccinated
jirds manifest increased lymphatic pathology and higher PGRN
after
challenge
uninfected
when
animals
intraperitoneal
compared
(Petit
infections,
et
to
normal
al.,
however,
L3
1993).
result
infected
or
Preexistent
in
lymphatic pathology after subcutaneous challenge
decreased
(Klei et
al., 1987) .
THE ROLE OF THE MACROPHAGE IN LYMPHATIC FILARIASIS
The macrophage (MAC) is implicated in the pathogenesis
of lymphatic
filariasis primarily because this cell is a
27
principal component of the granulomatous inflammation that
occurs in lymphatic and TPE lesions.
As an essential arm
and key cell of the immune response, the MAC may also play an
important effector and regulating role in the immune response
directed towards filariae.
The different filarial life cycle
stages have numerous opportunities to interact with the MAC
in diverse compartments of the monocyte phagocytic system;
L3 in the skin and lymph nodes,
lymph
nodes
and
granulomas,
granulomas and lymph nodes.
MF in the blood,
adults
and
spleen,
possibly
L4
in
At these sites, the MAC performs
its complex variety of functions
together with other immune
effector cells.
As L3 penetrate the mammalian host, the MAC of the skin
may
function
as
antigen
presenting
cell
(APC)
and
hypothetically may be able to direct infection into different
outcomes depending on genetic background of the host
Major histocompatibility complex,
natural
immunity,
(eg:
etc.)
The expression of surface molecules (eg: Mac-2, Receptor for
C3bi,
Fc
receptor)
vary
in
MACs
from
different
tissues
(Nibbering et al., 1985; Nibbering et al., 1987) which could
partly influence the presumed differences in host responses
to each of the filarial life cycle stages.
Thus, parasite-
MAC interactions may be crucial throughout the infection and
upon continuous reinfections driving the cascade of immune
responses that develop.
Filarial parasites are often found
covered by numerous inflammatory cells.
The MAC is the most
28
abundant (Soulsby, 1963; Jeska, 1969; Jeffers et al., 1987).
MACs have Fc and complement receptors (Lay and Nussenzweig,
1968) that mediate adherence to the parasite surface and may
be important in a larvacidal mechanism.
Rat MACs have been
implicated in cytotoxicity reactions against B. pahangi L3
(Chandrashekar et al., 1985) and B. malayi MF (Chandrashekar
et al., 1985; Chandrashekar
et al., 1986) in vitro.
MACs are known to be important producers of cytokines
and other molecules that function as mediators of immune and
inflammatory responses
1992).
1987;
Rappolee,
These products may have pro- or anti-inflammatory
effects.
TNF-of,
(Review in Nathan,
The
IL-1,
components,
metabolites,
factor,
and
ultimate
products that promote inflammation include
IL-6,
colony-stimulating factors,
coagulation
factors,
platelet-activating
fragments
of
arachidonic
factor,
fibronectin
complement
and
acid
platelet-growth
elastin.
objective of inflammation is destruction of
The
the
pathogen.
In order to fulfill this task the MAC must be
activated.
Adams and Hamilton (1988b) redefine the concept
of MAC activation as competence to mediate or complete a
complex
function
such as
processing
antigen or killing of microorganisms.
and presentation
They
of
distinguish
activation from capacities or attributes that can be measured
immunologically or biochemically such as number or affinity
of a certain receptors or secretion of a particular molecules
(Adams and Hamilton, 1988a).
29
More than one signal is required to fully activate MACs.
In the murine model, a priming signal, IFN-y and a triggering
signal,
TNF-a or LPS
are necessary
(Adams and Hamilton,
1987) . Signals that suppress MAC activation are less studied
but include TGF-/3 (Tsunawaki et al.,
1988),
al., 1989; Gautam et al., 1992), IL-10
1991; Gazzinelli et al.,
1992),
IL-4
(Liew et
(Mosmann and Moore,
prostaglandins
(review in
Schultz, 1991) and a2-macroglobulin-protease complexes (Adams
and Hamilton,
1988a; Crawford et al., 1994).
The two main
mechanisms that mediate parasite killing are the L-argininedependent nitric oxide intermediate
oxygen intermediate (ROI) pathways.
(NOI) and the reactive
Mouse MACs stimulated
with IFN-y and TNF secrete large amounts of NO (Ding et al.,
1988).
NO production seems to be the principal effector
mechanism in many murine systems of MAC-mediated microbial
killing (Green et al., 1991).
However, few studies have been
reported on the role of NO in cell-mediated helminth killing.
In
the
Schistosoma-mouse
model,
activated
schistosomula thru the production of NO
1989).
On
the
other
hand,
ROI
kill
(James and Glaven,
released
stimulated thru Fc receptors do not affect
viability (Scott et al., 1985).
MACs
by
phagocytes
schistosomula
In some systems, however,
ROI has been proved to be important.
known to be highly sensitive to ROI.
Filarial parasites are
Particularly,
hydrogen
peroxide has been found to be the most toxic oxygen species
for
the
1990).
filaria
Onchocerca
cervicalis
(Callahan
et
al,,
30
Schistosomula are killed by murine (James and Glaven,
1989)
and human
activated by
(Cottrell
lymphokines
et
al.,
1989)
and resistance
MACs
in
the mouse
associated with production of Thl cytokines,
IPN-y
(Smythies et al.,
1992).
previously
is
particularly
Thl and Th2
subsets are
characterized by the secretion of a contrasting and crossinhibitory array of cytokines.
Thl cells produce IFN-y, IL-2
and TNF-jS which are proinflammatory cytokines and promote MAC
activation.
involved
Th2
in B
secrete
cell
and
IL-10,
IL-4
eosinophil
and
IL-5 which are
development,
antibody
production and MAC deactivation (Mosmann and Coffman, 1989) .
Interactions
of
MACs
and
eosinophils
appear
important in parasitic and allergic conditions.
to
be
MACs release
eosinophil chemotactic and activating factors (Burke et a l .,
1991) . MACs secrete eosinophil cytotoxicity-enhancing factor
which primes eosinophils for enhanced cytotoxicity against
schistosomules
(Elsas et al.,
1987;
Lenzi et al.,
1985).
Conversely,
several studies (Haque et al., 1982; Nogueira et
al.,
Chandrashekar
1982;
et
al.,
1986)
have
found
that
eosinophil granules and other products are chemotactic for
MACs and enhance MAC cytotoxicity for nematode larvae.
Although the mechanisms of action of DEC are still uncertain,
DEC-mediated clearance of MF is thought to be dependent on
humoral
and/or
cellular
factors.
Several
studies
implicated the MAC in the destruction of MF during DEC
have
31
treatment and it has been proposed that initial DEC damage of
MF may trigger MAC-mediated cytotoxic mechanisms (Hawking et
al., 1950; Hayashi et al., 1983).
MACs
can
release
many
cytokines
fibroblasts and promote fibrosis,
growth factor
(IGF-1)
(PDGF), TNF-a,
TGF-/3,
fibroblast
that
stimulate
such as platelet-derived
insulin-like growth factor-1
growth
factor
(FGF) ,
colony
stimulating factor 1 (CSF-1), IL-1-0 and fibronectin (Review
in Kovacs, 1991).
Although the major mechanisms responsible
for the exuberant fibrous tissue deposition observed in some
elephantiasis
probably
degrees
patients
involved,
of
fibrosis
are
uncertain,
especially
that
in
MAC
promoting
generally
ensues
products
the
are
varying
granulomatous
inflammation.
The role that the MAC may play in lesion initiation,
persistence or termination during filarial infection may be
of central importance to understand disease pathogenesis.
More work is needed to clarify the relevance of the MAC to
immune and inflammatory responses in filariasis.
THE IMPACT OF DIFFERENT LIFE CYCLE STAGES ON THE IMMUNE AND
INFLAMMATORY RESPONSE
The
different
filarial
developmental
stages
are
morphologically, antigenically and biochemically different.
They are unlikely to induce an identical immune response in
the definitive host.
The ability of an individual's immune
system to cope with each of the life cycle stages may partly
32
contribute to the different outcomes of infection.
It is
also possible that each stage of the life cycle may have an
effect
on
the
immune
response
to
subsequent
stages
or
infections.
MF have been implicated as a likely candidate to induce
immunosuppression in filariasis.
Evidence for this exists in
both humans and experimental animal models.
Microfilarial
extracts from B. malayi have been found to suppress mitogendriven
lymphocyte
individuals
1987) .
proliferative
responses
and human filariasis patients
Treatment
with
DEC or
of
normal
(Wadee et
ivermectin
clears
MF
al.,
and
restores the immune responsiveness to filarial antigens in
most
individuals
1992).
However,
(Piessens
et al.,
1981;
ivermectin treatment
in
Lammie
jirds
et
does
al.,
not
restore proliferative cell responses or the PGRN (Brosshardt
et al., 1995) indicating that adult worms may be implicated
in filarial-induced hyporesponsiveness. Maizels and Lawrence
(1991) argue that both MF and adult parasites induce a state
of
immune
tolerance
that
allows
parasite
survival
and
prevents progression to disease.
Cellular and humoral immune responses against different
parasite stages have been studied primarily in the mouse
model.
Lawrence et al. (1994) observed contrasting cytokine
and Ig isotype responses induced by MF versus
long-lived
adult worms inoculated intraperitoneally in BALB/c mice.
MF
infection resulted in strong INF-y response that remained
33
elevated throughout the 28 days of the experiment.
IgGl,
IgG2a, IgG2b, and IgG3 levels were high throughout infection
and there was no increase
in IgE.
Female worms
stronger Th2 cell responses that male worms.
induced
IFN-y and IL-2
were not detected, but IL-4 was present in high levels and
was found to be produced by CD4 + T cells. IgE and IgGl were
also elevated.
These results indicate than adult worms,
particularly females, exert a strong and rapid polarization
of the immune response towards a Th2 phenotype, modulating
the Thl response induced by MF alone (Lawrence et al., 1994) .
Similar results have been observed by Pearlman et al.
(1993a)
during
infection.
early
However,
time
periods
these
of
authors
intraperitoneal
found
that
MF
prolonged
exposure to MF also results in a Th2 response.
It
is known
circumstances,
inflammatory
lymphatic
that
under
such as TPE,
stimulus.
vessels
has
certain not well
MF appear to act as a potent
However,
been
understood
pathological
primarily
damage
attributed
to
of
the
presence of adult worms (Von Lichtenberg, 1987/ Jungmann et
al.,
1992),
rather
than
to
MF.
Nevertheless,
histopathological studies in the jird (Vincent et al., 1980/
Klei et al., 1982/ Jeffers et al., 1987), ferret (Crandall et
al., 1982) and observations in humans (Meyers et al., 1976)
have found MF within lymphatic granulomas,
indicating that
this stage may initiate or enhance inflammatory reactions in
lymphatics and surrounding tissues.
34
MF have been shown to release prostaglandins E2 (Liu et
al., 1992) and other studies have stablished that MF of B.
malayi incorporate exogenous arachidonic acid (Longworth et
al., 1985).
PGE2 and prostacyclins are vasodilators, highly
active inhibitors of platelet aggregation and endothelial
adhesion, and potent immune modulators of leukocyte responses
(Taffet and Russell, 1980; Snyder et al., 1982; Ellner and
Spagnuold, 1986) . Secretion of prostanoid products by MF may
help
the
parasite
to
circulate
bloodstream (Liu et al., 1992).
intact
throughout
the
PGE2 has been reported to
selectively inhibit IgE and enhance IgG4 synthesis from human
B
lymphocytes
(Kimata et al.,
1991).
IgG4
may be very
important in parasitic infections preventing immunopathology
by several suppressive mechanisms (Hussain et al., 1987).
It
has been postulated that DEC works by altering arachidonic
acid
metabolism
entrapment
of
in
the
MF
and
in
host
parasite
in
the
cells
resulting
microvasculature
in
and
initiation of inflammatory reactions (Liu and Weller, 1990;
Kanesa-Thasan et al., 1991; Maizels and Denham, 1992).
In a study using immunoblotting of sera from endemic
normals and hyporesponsive microfilaremic individuals, it was
found that a 43 kD antigen derived from infective larvae was
preferentially
recognized
(Freedman et al.,
1989).
by
endemic
normal
individuals
Cloning and characterization of
this molecule has revealed that it is present primarily in L3
and MF, but not in adult worms, suggesting that larval stages
35
may
be
important
in
induction
of
protective
immunity
(Raghavan et al., 1994).
OBJECTIVES
Progress
lymphatic
models.
in
understanding
filariasis
is
the
immune
dependent
on
regulation
laboratory
The jird possesses distinct advantages
purpose.
Parasitological,
pathological
in
animal
for this
and immunological
events during B. pahangi infection have been well defined in
the jird and many aspects of the infection in this model
mimic
the
human
disease.
The
relatively
inexpensive
availability of the jird as inbred rodent along with the
recent
development
of
immunological
reagents
and
assays
enhances the usefulness of this animal model for the study of
filariasis.
The overall objective of the research described in this
dissertation
is
to
further
define
the
role
of
specific
parasite stages in the granulomatous inflammatory response
induced by B. pahangi.
The specific objectives are as follow:
Objective 1
To determine and compare the kinetics of the systemic
and peritoneal inflammatory responses during intraperitoneal
infection
of
B.
pahangi
evaluating
specificity of these responses.
the
parasite
stage
36
Objective 2
To determine the effect of low and high parasite burdens
of different parasite stages in the peritoneal cavity on the
systemic and peritoneal inflammatory response.
Objective 3
To
compare
the peritoneal
and systemic
inflammatory
responses induced by parasites irradiated with increasing
doses of gamma radiation, evaluating the effect of parasite
development on the induction of inflammatory responses.
Objective 4
To determine the state of macrophage activation during
the
course
of
parasite stages.
intraperitoneal
infections
with
different
CHAPTER 2.
DIFFERENTIAL INDUCTION AND REGULATION OF
INFLAMMATORY RESPONSES BY LIFE CYCLE STAGES
OF BRUGIA PAHANGI.
INTRODUCTION
Infection with the human lymphatic dwelling filarial
parasites
Wucheraria bancrofti,
Brugia malayi,
and Brugia
timori leads to a complex spectrum of clinical outcomes that
have
been
related
to
polarized
responses (Ottesen, 1980).
filarial-specific
immune
These immune responses range from
a complete hyporesponsiveness to parasite antigen which is
seen in most asymptomatic microfilaremic individuals to the
hyperresponsiveness observed in individuals with lymphatic
pathology and tropical pulmonary eosinophilia.
Individuals
who are equally exposed to infected mosquitoes but show no
signs of infection, so called "endemic normals", are presumed
to
have
developed
a protective
immunity
(Ottesen,
1980;
Ottesen, 1992; Nutman, 1995).
The
parasite
manifestations
outcomes
and
host
factors
and progression of
are not well understood.
that
determine
the different
However,
the
clinical
it has been
hypothesized that the various developmental stages of the
parasite
induce
responsible
for
different
these
responses
diverse
(Maizels and Lawrence, 1991).
and
clinical
are
in
part
manifestations
Microfilariae (MF) have been
implicated as an important determinant in the state of immune
hyporesponsiness observed in most infected individuals who
are
asymptomatic.
The
vast
37
majority
of
microfilaremic
38
asymptomatic
patients
exhibit
suppressed
B
and
T
cell
responses to filarial antigen (Nutman et al., 1987b; King et
al., 1992; King et al., 1993).
diethylcarbamazine
1988)
and
(Piessens et al.,
ivermectin
responsiveness
The microfilaricidal drugs
in
(Lammie
the
1981;
et
majority
Lammie et al.,
al.,
of
1992)
the
restore
microfilaremic
asymptomatic individuals.
Conversely, inflammatory changes
in the
lymphatic vessels
are mostly attributed
immune
responses
directed
towards
adult
to
local
worms
(Von
Lichtenberg, 1987; Jungmann et al., 1992; Nutman, 1995) and
protective immunity has been hypothesized to be targeted to
early larval stages, primarily L3 (Day, 1991).
The
pahangi
jird
is
(Mongolian
an
downregulation
useful
of
the
unguiculatus)
infected
model
to
study
immune
response.
with
B.
filaria-mediated
Jirds
develop
persistent microfilaremia associated with a state of immune
hyporesponsiveness similar to that observed in microfilaremic
asymptomatic
filarial
human patients.
specific
In
the
responsiveness
is
jird,
the
loss
characterized
of
by
decreased antigen-induced proliferative responses of spleen
cells (Lammie and Stephen, 1983; Klei et al., 1990) and renal
lymph node cells
(Rao et al., 1995), and downregulation of
granulomatous responses around antigen-coated beads embolized
in the lungs (PGRN)
However,
(Klei et al., 1981; Klei et al., 1990).
maintenance
dependent on MF,
of
the
hyporesponsive
state
is
not
since jirds with occult infections still
39
show downregulation and treatment of microfilaremic jirds
with
ivermectin
removes
MF
but does not
restore
immune
responsiveness (Brosshardt et al., 1995; Lin et al., 1995).
In the jird model, the pathologic and immunologic events
that occur in the host after a primary subcutaneous infection
have been defined.
Although the chronology of the filarial
life cycle in the jird is well known,
the contribution of
each
stage
specific
filarial
developmental
responses is still not clear.
to
the
host
The objective of the present
chapter is to better discern the influences that different
life
cycle
stages
of
B.
inflammatory responses.
pahangi
Brugia
may
have
8pp. have
in
been
the
host
shown
to
develop normally and induce systemic immune responses when
implanted into the peritoneal cavity (McCall et al.,
1973;
Klei et al., 1981; Jeffers et al., 1987; Klei et al., 1987).
Further,
all
stages
found in the vertebrate
host
can be
recovered from this site and transplanted into other animals.
This
transplantation
interactions
and
system
kinetics
was used to
ofparasite
study worm-cell
stage-induced
inflammation.
MATERIAL AMD METHODS
Animals
Each treatment group consisted of inbred, 6 to 8 week
old, male, Mongolian jirds (Meriones unguiculatue) . Animals
were obtained from Tumblebrook Farms
(West Brookfield, MA)
and were maintained on standard rodent chow and water ad
libitum.
40
Parasites
B.
pahangi
L3
were harvested
by
crushing
cold-
anesthetized Aedes aegypti followed by sedimentation in a
Baermann apparatus
1990).
as previously described
(Klei et al.,
4th stage larvae were obtained from the peritoneal
cavities of jirds that had been infected with L3 for a period
of
14
days.
Mature
adult
male
and
female
worms
were
harvested from jirds with patent (> 60 DPI) intraperitoneal
(IP)
by
repeated
peritoneal lavages with phosphate buffered saline
(PBS) pH
7.4.
infections.
Worms
Parasites
were
sexed,
were
collected
washed
in
HEPES
hydroxyethylpiperazine-N'-2-ethanesulfonic
acid)
RPMI-1640
NY)
medium
penicillin
(GIBCO,
Grand
containing
containing 2 to 3 ml of
and
fresh
Single sex inoculations were performed using 16
gauge needles.
MF obtained from peritoneal washes of jirds
with chronic patent
density
buffered
(100 units/ml) and streptomycin (100 ug/ml)
transferred into syringes
medium.
Island,
(N-2-
infections were
centrifugation
separated by Percoll
(Chandrashekar
et
al.,
1984)
and
filtration through a 25 mm diameter 8 /zm Nucleopore membrane
(Millipore Corporation,
al.,
1975).
method.
Bedford,
MA 01730)
MF could not be separated from
Therefore,
both MF and
infections.
Dead
adult
experiment.
Mature
eggs by
eggs wereused
parasites
worms
(Ponnudurai et
were
recovered
used
from
the
in
in
this
the
another
peritoneal
cavity were killed by 5 successive cycles of rapid freezing
and thawing.
41
Parasite antigen:
Adult worms were collected from the peritoneal cavity of
infected patent jirds and antigen extracts were prepared as
previously described (Klei et al., 1988).
mature
adult
female
and
male
B.
Briefly, frozen,
pahangi
were
cut
and
disrupted in a Tenbroke tissue grinder in PBS containing 2.5%
Aprotinin
(Sigma Chemical Co.,
St.
Louis,
MO).
Parasite
soluble extracts (SAWA) were filter sterilized and stored at
- 70°C.
Lung granulomas
A pulmonary granulomatous response (PGRN) was induced by
the embolization of Cyanogen bromide Sepharose 4B (CNBr-4B)
beads
(Sigma Chemical Co,
previously described
St. Louis,
(Klei et al.,
MO)
in the lungs as
1988).
Briefly,
sized
CNBr-4B beads were coated with SAWA or with diethanolamine
(DEA)
(Sigma), and were inoculated into the retro-orbital
sinus 3 days prior to necropsy.
Each jird received 2.5 x 104
beads diluted in 0.5 ml of 0.05% saline.
At necropsy, lungs
were perfused with 10% formalin via the trachea.
Tissues
were
2.5 /xm.
embedded in paraffin and step sectioned at
Several sections from each lung were cut at 50 /xm intervals
and were stained with hematoxylin and eosin.
Areas of 20
granulomas around 40 to 60 um diameter beads were measured in
each animal
using a
image analysis
Biometrics, Nashville, TN).
system
(Bioquant
IV,
42
Samples collected:
Animals were bled from the retro-orbital plexus prior to
euthanasia.
MP counts were determined in 20 ul of fresh
blood.
peritoneal
The
extensively with PBS.
cavity
of
each
jird
cell counts were determined
Electronics,
peritoneal
samples
washed
Peritoneal washes were collected and
cells were concentrated by centrifugation.
(Coulter
was
in a automated counter, model ZF
Inc,
1:500
Total peritoneal
in
Hialeah,
Isoton
FL)
II
after
diluting
coulter
balanced
electrolyte solution (Coulter Corporation, Miami, FL).
Two
cytospin preparations were made of each sample and stained
with
either
a
modified
Giemsa
stain
esterase (NSE) stain (Yam et al., 1971).
to
obtain
a
more
macrophage numbers.
accurate
or
a
non-specific
NSE stain was used
measurement
of
peritoneal
MF were counted in 20 ul of individual
peritoneal wash concentrated to 1 ml and are expressed as the
mean
MF
per
abdominal
searched for worms.
cavity.
Peritoneal
washes
were
Carcasses were soaked in PBS for at
least 1 hour to recover remaining worms.
Statistical Analysis:
Significant differences between group means were tested
with
a
comparative
analysis
Studentized Range test.
of
variance
using
Tukey's
Results were considered significant
when P values < 0.05 were obtained.
43
RESULTS
Kinetics o£ the P6RN in jirds inoculated intraperitoneally
with L3.
All previous studies utilizing PGRN to measure systemic
granulomatous
inflammation to parasite
antigen
have
been
conducted in jirds infected via the subcutaneous route (Klei
et al., 1990).
Following this procedure parasites develop
primarily in the lymphatics
(Ash, 1973) .
The
purpose of
this experiment was to demonstrate that the kinetics of the
PGRN in intraperitoneal (IP) infections with L3 is similar to
that described following subcutaneous infections (SC).
groups of 24 animals were used.
Two
One group was inoculated IP
with 75 L3 and the other group received medium IP containing
mosquito debris recovered from L3 harvest.
Necropsies were
performed at 7, 28, 56 and 181 days post-infection (DPI).
The granulomatous response induced in the lungs by SAWAcoated beads (PGRN) in the L3 infected group peaked at 28 DPI
(P < 0.005)
and decreased progressively after that point
(Fig.2.1.).
At 181 DPI PGRN of L3 infected animals was not
significantly different from the PGRN of uninfected animals
that
received
antigen-coated beads,
or
from
infected
uninfected animals that received DEA-coated beads.
or
Control
animals receiving DEA- or antigen-coated beads had, at any
time period, granuloma areas less than 7,000 /un2.
The macrophage
(MAC) was the cell that accumulated in
higher numbers in the peritoneal cavity of L3 infected
44
El 7 DPI
■
28 DPI
ffl 56 DPI
■
L3
181 DPI
Control
T reatm ent G roups
Figure 2.1.
Pulmonary granulomatous response to Brugia
pahangi antigen-coated beads in jirds inoculated IP with L 3 .
PGRN of infected and uninfected animals to DEA-coated beads
is not shown, but was not significantly different from PGRN
of uninfected jirds to B. pahangi antigen represented as
control in this figure or from PGRN of L3 infected jirds to
antigen at 181 DPI (P < 0.05).
PGRN is measured as /zm2.
Bars represent ± 1 standard deviation of the mean.
45
animals and was used as primary measurement of the peritoneal
inflammatory response.
Other cell types observed in the
cytospins
of
included
eosinophils,
neutrophils.
peritoneal
lavages
from
lymphocytes,
infected
mast
In the L3 infected animals,
animals
cells
and
peritoneal MACs
were elevated throughout the infection period and peaked at
56
and
181
eosinophils
DPI
(P
<
0.05)
(Fig.
increased markedly after
elevated throughout the infection
2.2.).
7 DPI
Peritoneal
and
(P < 0.05).
remained
Peritoneal
eosinophils in control animals were absent or present
minimal numbers (Fig. 2.3.).
L3
infection increased at
in
Peritoneal lymphocytes in the
28 and 56 DPI
decreased to levels of controls at 181 DPI.
(P < 0.05)
and
Peritoneal mast
cells from infected animals did not vary significantly from
controls.
Peritoneal
neutrophils
animals at the acute time periods.
increased
in
infected
A few, small (about 1 mm
in diameter), peritoneal granulomas were found covering the
serosal
surface
of
liver and
stomach in
some
of
the
L3
infected jirds at 181 DPI.
Parasite recovery decreased throughout infection until
56 DPI.
In the L3 infected group, MF were present in large
numbers in the abdominal lavages at 56 and 181 DPI
2.1).
(Table
4 out of 6 animals infected with normal L3 had MF in
the peripheral blood at 181 DPI.
46
m
%
7 DPI
28 DPI
CA
d
w
U
0
56 DPI
■
181 DPI
w
§
W
L3
Control
T reatm ent G roups
Figure 2.2.
Peritoneal macrophages in jirds inoculated IP
with L3 B. pahangi. Macrophages are expressed as mean number
of non-specific esterase (NSE) positive cells per peritoneal
cavity. Note the increase of NSE + cells as infection with
L3 progressed. Greatest number of macrophages accumulated at
56 and 181 DPI in the L3 infection.
Low numbers of
macrophages were recovered from uninfected animals throughout
the infection. Bars represent the standard deviation of the
mean.
47
12.5i
ffl 7 DPI
M
CO
10-
eu
O
5
CO
O
W
K
■
28 DPI
^
56 DPI
■
181 DPI
7.5-
2.5-
QJUC
1--Control
T reatm ent G roups
Figure 2.3.
Peritoneal eosinophils in jirds inoculated IP
with L3 B. pahangi. Eosinophils are expressed as mean number
per peritoneal cavity. Number of eosinophils in L3 infected
animals are markedly increased after 7 DPI when compared with
controls. Bars represent standard deviation of the mean.
48
Table 2.1.
Worm Recovery and microfilaria counts in
the peritoneal cavity of jirds inoculated IP with L3a.
DAYS
POST INFECTION
7 DPI
WORM
RECOVERY
PERITONEAL
MICROFILARIAE
16.5 ± 7.97
-
28 DPI
15.5 ± 10.56
-
56 DPI
4.5 ± 3.33
29175 ± 21936
181 DPI
9.7 ± 5.71
354528 ± 210411
Results are expressed as mean ± standard deviation.
49
Comparison of the peritoneal and systemic inflammatory
response in jirds inoculated with L4, male or female adult
worms.
The hypohtesis that downregulation of the PGRN is not
dependent on MF was tested by comparing the PGRN response to
antigen-coated beads in jirds receiving infections of female
worms which produced MF to that of male worms.
PGRN to
parasite antigen was also investigated in jirds inoculated
with L 4 ; in order to compare the effect of this pre-adult
stage on the kinetics of the inflammatory response with that
induced
by
inflammatory
infections
response
of
adult
induced
parasites.
by
the
Peritoneal
different
parasite
stages was also evaluated.
Four groups of 40 jirds each was
inoculated IP with 26 L4,
12 adult male,
worms
or medium,
respectively.
inflammatory response
to
these
The
12 adult female
PGRN and peritoneal
infections were
compared.
Necropsies were performed at 7, 14, 28, 56 and 166 DPI.
The PGRN in the female and male worm infected animals
was elevated at 7 and 14 DPI.
In the L4 infected group
granuloma areas at 7 DPI were significantly smaller
(P <
0.005) than those induced by the female or male infection at
the same time period (Fig. 2.4.), but increased markedly at
14 DPI.
After 14 DPI there was a trend of PGRN decrease in
all infected groups.
infected
groups
was
At 56 and 166 DPI the PGRN of all
markedly
significantly different (P <
donwregulated
and
not
0.05) from the PGRN of infected
50
7 DPI
14 DPI
"e
0
28 DPI
□
56 DPI
9
K
166 DPI
<
■
o
Male
Female
Control
Treatment Groups
Figure 2.4.
Pulmonary granulomatous response to parasite
antigen in jirds inoculated IP with male, female or L4 B.
pahangi.
Granuloma areas are expressed in /un2. Note the
downregulation of the granulomatous response in all the
infected groups at the chronic time periods (56 to 166 DPI).
Bars represent one standard deviation of the mean.
51
animals to DEA or from the PGRN of uninfected animals to DEA
and antigen.
Furthermore, PGRN at 56 was not significantly
different from the PGRN at 166 DPI in any group.
MACs
accumulated
in
large
cavities of all infected jirds.
numbers
in
the
abdominal
However, differences were
seen in the kinetics and numbers of cells present.
female
worm
infected
group
MAC
numbers
In the
increased
above
controls after 7 DPI and peaked at 28 DPI remaining elevated
throughout the experiment (P < 0.001)
infected
group
peritoneal
MAC
(Fig. 2.5.) . In the L4
numbers
increased
above
controls after 7 DPI and continued to increase to become
highest at 166 DPI
(P < 0.05).
In the male worm infected
group, MACs were elevated at 28 and 56 DPI (P < 0.005), and
in
the
remaining
time
periods
their
different from uninfected controls.
numbers
were
not
Peritoneal eosinophils
peaked at 56 DPI in the adult female worm (P < 0.001) and L4
(P < 0.05) infected groups (Fig. 2.6.).
In the female worm
infection peritoneal eosinophils decreased significantly (P
< 0.001) at 166 DPI.
Peritoneal eosinophils in the male worm
infection increased above controls only at 28 DPI (P < 0.05) .
Granuloma formation on peritoneal surfaces was not observed
grossly in any of the animals.
Female worm recovery decreased after 7 and 14 DPI (P <
0.05) and did not differ significantly thereafter.
L4 and
male worm recovery did not differ significantly throughout
infection (Table 2.2.).
52
40
E l 7 DPI
■
14 DPI
0
28 DPI
□
56 DPI
■
166 DPI
X
Vi
W
O
<
seu
O
os
u
30-
Tl
W
Pu
Female
Control
Treatment Groups
Figure 2.5.
Peritoneal macrophages in jirds inoculated with
male,
female or L4 B. pahangi.
Greatest macrophage
accumulation is present in animals inoculated with either L4
or female worms.
Bars represent standard deviation of the
mean.
53
20E3 7 DPI
co
15-
oO
S
CO
O
W
■
14 DPI
0
28 DPI
□
56 DPI
■
166 DPI
10-
5-
B
Female
Control
Treatment Groups
Figure 2.6.
Peritoneal eosinophils in jirds inoculated with
male,
female or L4 B. pahangi.
Greatest eosinophil
accumulation is induced by L4 and female worms.
Bars
indicate standard deviation of the mean.
Table 2.2. Parasite recovery in jirds inoculated with
male, female or L4 B. pahangi
DAYS
POSTINFECTION
HALE WORM
INFECTION
FEMALE WORM
INFECTION
L4
7 DPI
5.88 ± 3.95
9 ± 1.22
8.25 ± 5.7
14 DPI
3.13 ± 2.66
6.13 ± 2.5
4.4 ± 2.12
28 DPI
4.63 ± 3.57
5.6 ± 2.8
1.86,± 1.8
56 DPI
2.5 ± 2.29
166 DPI
3.14 ± 2.6
3.9 ± 2
1.12 ± 0.8
a Results are expressed as mean ± standard deviation.
3.6 ± 4.9
5 + 2
55
Microfilariae were present in the abdominal cavity at
all time periods in the adult female infected group, peaking
at 56 DPI
(P < 0.005) . In the L4 infection, peritoneal MF
were found only at 56 and 166 DPI
(Fig. 2.7).
No MF were
present in peripheral blood in any of the infected groups at
any of the necropsy time periods.
Effect
of
The
dead
worms
hypothesis
downregulate
the
on
that
the
granulomatous
living
granulomatous
response.
worms
are
needed
to
response
was
tested
by
comparing the PGRN of jirds inoculated with dead worms to
that of
jirds
parasites.
inoculated with the same number of viable
Two groups of 15 animals were inoculated IP with
5 dead female worms and 5 dead male worms or with the same
number
and
sex
of
living
inoculated with medium.
worms.
A
control
group
was
Necropsies were performed on 5
animals per group at 15, 57 and 108 DPI.
Animals inoculated with dead worms had elevated PGRN to
SAWA-coated beads at all time periods.
Animals that were
infected with living worms had at 15 DPI large granuloma
areas not significantly different from dead worm inoculated
animals.
Granuloma areas decreased progressively in the
living worm inoculated animals to become similar to control
animals
at 108 DPI (Fig. 2.8.).
56
250
Female
L4
200-
150
100-
7
14
28
166
DPI
Figure 2.7.
Peritoneal microfilaria counts in jirds
inoculated with female worms or L 4 . MF are expressed as
total number per peritoneal cavity.
57
40
■
IS DPI
EE 57 DPI
■
Dead
Living
108 DPI
Control
T reatm ent G roups
Figure 2.8.
Pulmonary granulomatous response to parasite
antigen in jirds inoculated with dead or living worms.
Downregulation of the granulomatous response was induced by
living but not dead worms. Granuloma areas are expressed in
Hm2. Bars represent standard deviation of the mean.
58
Effect of parasite number on the peritoneal and systemic
inflammatory responses.
The
effect
of
different
parasite
burdens
on
the
peritoneal and systemic inflammatory responses was tested.
Jirds in four groups of 20 animals each were infected IP with
30 adult male worms,
5 adult male worms,
worms or 5 adult female worms.
inoculated with medium.
56 and
167 DPI.
30 adult female
Jirds in a control group were
Necropsies were performed at 7, 14,
15 animals
in another group were
each
inoculated IP With a mixture of 10,000 MF and 27,000 eggs.
Necropsies for this latter group were performed at 7, 14 and
56 DPI.
Pulmonary granuloma areas induced by SAWA-coated beads
in
animals
inoculated with MF,
30
adult
male,
30
adult
female, 5 adult male, or 5 adult female worms were greatest
at 7 and 14 DPI
(P < 0.05)
(Figs 2.9. and 2.10).
At these
early time periods the PGRN of groups inoculated with 30
female worms was less than that of the group inoculated with
5 female worms
periods,
(P < 0.007)
however,
(Fig. 2.9.).
At chronic time
the parasite burden did not effect the
level of PGRN downregulation.
The PGRN induced by 5 or 30
female worms at 56 DPI was not significantly different from
controls.
However,
PGRN induced by MF,
5 male or 30 male
worms at 56 DPI was greater than the PGRN of controls
(P <
0.01) . Different doses of male worms did not effect the PGRN
(Fig. 2.10.).
MAC numbers
in the peritoneal
cavity increased more
pronouncedly throughout the infection in the animals that
59
80
0
7 DPI
14 DPI
□
56 DPI
■
167 DPI
30 Females
5 Females
PARASITES IMPLANTED
Figure 2.9.
Comparison of the pulmonary granulomatous
response induced in jirds with IP inoculation of high and low
numbers of female worms as well as MF.
30 female worms
induced significantly smaller granuloma areas than 5 female
worms at 7 and 14 DPI (P < 0.05) . Downmodulation of the PGRN
occurred at 56 and 167 DPI in animals inoculated with
different numbers of female worms or at 56 DPI in animals
inoculated with MF.
Granuloma areas are measured in /zm2.
Bars represent standard deviation of the mean.
60
□
7 DPI
14 DPI
"s
9
□
56 DPI
167 DPI
30 Males
5 Males
Control
PARASITES IMPLANTED
Figure 2.10.
Comparison of the pulmonary granulomatous
response in jirds inoculated IP with high and low male worm
numbers.
No significant difference in PGRN was found in
jirds inoculated with 30 or 5 numbers of male worms.
Bars
represent standard deviation of the mean.
61
received 30 adult female worms (data not shown). A few (2 or
3) macroscopic granulomas (about 1 mm in diameter) were found
on the serosal surface of liver and stomach, as well as in
the omentum of some of the animals inoculated with 30 female
worms at 56 and 167 DPI.
The parasite recovery of animals
female
adult
worms
was
significantly
inoculated with 30
greater
than
the
recovery of animals inoculated with 5 female worms only at 7
DPI
(P < 0.05)
animals
(Table
2.3.).
inoculated with
30
The parasite
male
higher than in animals inoculated
worms
was
recovery of
significantly
with 5 male worms at all
time periods (P < 0.05).
Peritoneal MF counts in the 30 female worm infection
were significantly increased over those in the 5 female worm
infection at 56 and 167 DPI ( P < 0.05), but not at 7 or 14
DPI.
The MF recovery in jirds inoculated with MF was very
low and significantly decreased over time when compared with
the MF recovery in the female worm infections (Fig. 2.11.).
DISCUSSION
Induction and maintenance of the hyporesponsive state in
the jird is not uniquely dependent on the presence of MF.
This
is
consistent
with
previous
indirect
(Brosshardt et al., 1995; Lin et al., 1995) .
study
all
life
cycle
stages
induced
an
observations
In the present
early
systemic
granulomatous response to parasite antigens as measured by
the
artificial
lung granuloma model.
PGRN response
was
downmodulated as infection progressed to the chronic time
Table 2.3.
Parasite recovery from jirds inoculated with
different numbers of adult worms*.
DAYS
POSTINFECTION
30 MALE
WORMS
5 MALE
WORMS
30 FEMALE
WORMS
5 FEMALE
WORMS
3 ± 1.67
23.7 ± 4.4
2.6 ± 1.5
7 DPI
20.4 ± 2.7
14 DPI
17.6 ± 7
4.4 ± 0.8
5.5 ± 2.5
1.6 ± 1.6
56 DPI
17 ± 6.27
3 ± 1.26
4.25 ± 3.9
0.6 ± 1.2
167 DPI
9.8 ± 8.3
3.2 ± 1.16
0.5 ± 0.5
Results are expressed as mean ± standard deviation.
0
63
300
30 Female
5 Female
MF
10050-
7 DPI
14 DPI
56 DPI
Figure 2.11.
Peritoneal microfilaria counts in jirds
inoculated with different female worm numbers or with MF.
Note the elevated numbers of MF present in jirds with
inoculations of 30 female worms.
64
period in the groups that were inoculated with male, female,
L4,
L3
or
MF.
responsible
for
This
the
suggests
that
induction of
the
parasite
factors
downregulation
are
present in multiple parasite stages.
Inoculation
granulomatous
remained
induce
dead
adult
downregulation
large
inoculation.
of
at
all
time
worms
and
did
pulmonary
.points
following
products
molecules
of
induce
granulomas
dead
worm
This indicates that living worms are needed to
a filarial-specific hyporesponsive
parasite
not
worm
relevant
to
state
and that
immunomodulation
metabolism.
A
number
may
of
be
these
immunomodulatory molecules have been described (Wadee et a l .,
1987; Lai et a l ., 1990).
Alternatively, it is possible that
inflammatory downmodulation requires constant and cumulative
release of parasite antigens which would only occur if the
worms are alive.
This would suggest that antigenic load
produced by chronicity is important in the state of immune
hyporesponsiveness.
Our results show that the degree of PGRN was modified by
different parasite burdens.
Significant difference in the
PGRN between inoculation of different female worm numbers was
found
early
periods.
in
infection,
but
not
at
the
chronic
time
No significant differences were found in groups
infected with different numbers of adult male worms, which
would suggest the existence of separate factors in male and
female worms which effect immunoregulation.
Alternatively,
65
it is possible that the female worms produce more of the same
immunoregulatory factors.
alone
induced
Interestingly, inoculation of MF
downregulation
of
the
PGRN
at
56
DPI.
Moreover, this dowmodulation was less pronounced than in the
female and male worm infected groups at the same time period.
The lighter antigenic load of MF inoculated in a single bolus
could again explain this difference.
The effect of different
MF burdens alone, however, was not tested.
the
B.
pahangi- infected
jird
have
Other studies in
found
significant
correlation between the degree of PGRN downregulation and the
levels of circulating MF and female worm recovery suggesting
that the parasite burden positively influences the degree of
hyporesponsiveness (Brosshardt et al., 1995).
(1993)
Petit et al.
found a correlation between the PGRN and levels of
circulating MF in jirds with similar adult worm burdens.
Immunological observations in human patients infected with
filariae have consistently found diminished parasite-specific
lymphocyte
proliferative
circulating MF when
responses
in
individuals
compared to amicrofilaremic
with
subjects
(Ottesen et al., 1977; Piessens et al., 1980; Nutman et al.,
1987a; King et al., 1992).
Studies of cytokine profiles have
shown that the asymptomatic microfilaremic group of patients
have
a
shift
toward
a
predominantly
Th2
response
that
contrast with the Thl-like response of chronic pathology
patients
(King
et
al.,
1993;
Maizels
et
al.,
1995).
Detection of cytokine mRNA in spleen and lymph nodes from
66
23. pahangi-infected jirds have demonstrated a pattern similar
to that found in human patients.
found
in
the
jird
mRNA for IL-10 and IL-4 are
coinciding
with
the
state
of
hyporesponsiveness that occurs as the infection progresses to
the chronic period
known
to
posses
properties
development
parasites.
1996).
These Th2 cytokines are
immunomodulatory
(Mosmann
of
(Mai,
and Moore,
pathology
and
and
1991)
favor
antiinflammatory
that
would
prevent
persistence
of
the
Experiments in the mouse model have demonstrated
that IP inoculated adult worms rapidly stimulate a Th2 cell
response, whereas MF induces a immediate Thl cell response
(Lawrence et al., 1994).
MF
and
repeated
However, prolonged IP exposure to
(but not
single)
SC
inoculations
of
MF
antigen also induce a Th2 phenotype (Pearlman et al., 1993a;
Pearlman et al.,
1993b).
The mechanisms
that drive
the
expansion of a Th2 response are not well understood,
but
chronicity and high load of antigen have been shown to be
important (Bretscher et al., 1992; Parish and Liew, 1972).
Other
factors
that
have been
implicated
different Th subsets include APC
costimulatory molecules
in
induction of
(Gajewski et al.,
(Weaver et al.,
1988),
1991),
Th subset-
specific antigen (Scott et al., 1988; Liew et al., 1990) and
the initial cytokine milieu (Seder et al., 1992).
Filarial
antigens may exist that activate preferentially specific Th2
subsets.
It is also possible that certain filarial molecules
trigger an initial release of Th2 cytokines by non T cell,
67
creating
the
cytokine
proliferation
cytokines.
and
milieu
appropriate
downregulation
of
for
Th2
proinflammatory
Our data suggest that if such immunomodulatory
molecules exist they are shared by various parasite stages.
Interestingly,
primarily
peritoneal
characterized
by
inflammatory
MAC
response,
accumulation,
did
not
correspond to the pulmonary granulomatous
response.
The
largest
present
the
numbers
of
peritoneal
MACs
were
at
chronic time period in infections with L4, adult female worms
or L 3 . This elevation in MAC numbers was associated with the
presence of large numbers of peritoneal MF and low pulmonary
granulomatous responses.
result
in
production
Conversely, infections that did not
of
numerous
viable
MF,
such
as
inoculations of adult male worms, MF or adult dead worms,
induced low levels of peritoneal MACs.
that
MF
may
be
a
potent
These data suggest
inflammatory
stimulus
when
compartmentalization phenomena occur, and result in localized
reactions independent of the systemic response..
The
magnitude
of
the
intraperitoneal
inflammatory
reaction and the feasibility of inoculating different life
cycle stages into the peritoneal cavity makes this site an
ideal body compartment to study cell-parasite interactions
during B.pahangi infection (Klei et al., 1981; McCall et al.,
1973; Jeffers et al., 1987).
are
comparable
to
Intraperitoneal inoculations
subcutaneous
infections
in
that
the
cellular composition of the inflammatory response caused by
68
B. pahangi is similar in both peritoneal cavity and lymphatic
vessels
Previous
(Jeffers
et
studies
al.,
have
1984;
also
Jeffers
shown
et
that
al.,
1987).
preexistent
intraperitoneal infections result in decreased granulomatous
lymphatic lesions after subcutaneous inoculation
al.,
1987).
(Klei et
Present results show that the PGRN to worm
antigen follows similar kinetics following L3 inoculation in
both body locations.
Inoculation of L3 IP can downregulate
the systemic inflammatory response in the same manner as L3
that localize in the lymphatic system (Klei et al., 1981; Rao
et al.,
1995).
This suggests that the IP implantation of
adult stages can also mimic specific responses that occur in
the natural location of filariae.
In conclusion, viable worms are necessary to induce and
maintain
the hyporesponsive
state,
responsible for this condition.
but
MF
alone
are
not
Our experiments also suggest
that parasite burden is important in downmodulation of the
inflammatory response.
Further investigations are necessary
to determine whether early larval
stages,
are
involved
in
the
granulomatous response in the jird.
address this question in chapter 3.
stages,
the L3
downregulation
and L4
of
the
Attempts were made to
CHAPTER 3.
EFFECT OF GAMMA RADIATION ON BRUGIA L3
DEVELOPMENT IN VIVO AND THE KINETICS OF
GRANULOMATOUS INFLAMMATION INDUCED BY THESE
PARASITES
INTRODUCTION
Lymphatic filariasis is a parasitic disease caused by
Wuchereria
bancrofti,
affects
120
million
regions
(WHO, 1992;
Brugia
people
malayi
in
and
tropical
B.
timori
and
that
subtropical
Ottesen and Ramachandran, 1995).
The
disease has a complex spectrum of clinical outcomes that have
been related to different immune responses of the host to
filarial
antigen
(Ottesen,
1980;
Piessens
et
al.,
Nutman et al., 1987a; Ottesen and Ramachandran,
endemic areas,
1980;
1995).
In
the majority of the infected population is
generally asymptomatic and microfilaremic.
Pathology occurs
in a smaller proportion of infected people, and interestingly
most
of
chronic
these
patients
lymphatic
disfiguring lesions.
individuals
in the
are
disease
amicrofilaremic.
often
results
in
However,
dramatic
A smaller and not well studied group of
filarial
spectrum
are
those
that
are
thought to be putatively immune; so called endemic normals.
The relatively polarized immune responses of microfilaremic
asymptomatic individuals versus amicrofilaremic symptomatic
patients have lead to the suggestion that the microfilaria
stage
is
responsible
for
the
immune
hyporesponsiveness
characteristic of most microfilaremic individuals.
Studies
in human and experimental animals have partly supported this
69
70
hypothesis (Piessens et al., 1981; Wadee et al., 1987; Lammie
et al., 1988; Lammie et al., 1992) . However, it is now clear
that MF is not the only life cycle stage involved in the
downregulation of the granulomatous response (Brosshardt et
al.,
1995;
however,
Lin et al.,
1995),
(Chapter 2).
Scant work,
has been done on the role that other life cycle
stages play in the diverse immune responses to filaria.
The
jird
(Meriones
unguiculatus)
extensively for the study of filariasis.
has
been
used
Jirds chronically
infected with B. pahangi develop an immune hyporesponsive
state (Klei et al., 1981; Lammie and Stephen, 1983; Klei et
al.,
1990)
that may be similar to that observed in human
filariasis
(Ottesen et al.,
Nutman et al.,
al.,
1992).
investigate
1977;
Piessens et al.,
1987a; Maizels and Lawrence,
This model
1991; King et
is particularly advantageous
the factors and mechanisms
events
have
the
parasitology,
been
well
The
pathology and immunologic
characterized
during
primary
subcutaneous infections with B. pahangi in male jirds.
timing of
to
implicated in the
induction and maintenance of this hyporesponsive state.
kinetics of
1980;
The
these events can be correlated to the periods of
parasite development.
An in vivo pulmonary granuloma model
has been used to measure inflammatory responses induced by
parasite
antigen
(Klei
et
al.,
1981).
Pulmonary
granulomatous responses (PGRN) to soluble adult worm antigen
(SAWA)
peak at 14 days postinfection
(DPI)
and begin to
71
decrease before patency (56 DPI), when adult worms are still
immature (Rao et al., 1995).
Proliferative responses to SAWA
of renal lymph node cells draining the site of infection
follow kinetics that are similar to the PGRN
1995).
Spleen
cell
blastogenesis
subsequently decreases.
(Rao et a l .,
increases
later
Lymphatic granulomas peak between 60
and 90 DPI and decrease significantly thereafter
al.,
1988;
Klei
inflammatory
et
al.,
responses
and
1990).
in
B.
Depressed
(Klei et
immune
and
jirds
are
pahangi-infected
maintained throughout the chronic period in association with
a persistent microfilaremia.
However, a small percentage of
chronically
are
infected
jirds
amicrofilaremic.
These,
however, are not resistant to reinfection and do not respond
to
antigen
indicating
that
the
hyporesponsive
state
is
maintained in the absence of MF (Brosshardt et al., 1995; Lin
et al., 1995).
with
Furthermore, chemotherapeutic removal of MF
ivermectin
in
jirds
does
not
restore
immune
responsiveness (Brosshardt et al., 1995).
Intraperitoneal
jird
mimic
(IP)
subcutaneous
infections of J9. pahangi in the
infections
in
components of the inflammatory response
that
the
cell
(Jeffers et al.,
1987) and the kinetics of the PGRN are similar (Chapter 2).
Parasite inoculation into the jird peritoneal cavity at any
stage of development remain in this site, continue to develop
normally and can easily be recovered (Malone et al., 1974).
Thus,
the
peritoneal
cavity
has
been
used
as
an
ideal
72
compartment to study the effect of different filarial life
cycle
stages on the peritoneal and systemic inflammatory
responses.
worms
These experiments have demonstrated that viable
are
necessary
to
initiate
and
maintain
the
downregulation of the PGRN induced by SAWA-coated beads, and
that this downregulated state is not dependent on MF (Chapter
2) .
Gamma
radiation
is
known
to
interrupt
development of nematodes and this effect
radiation dosage (Oothuman et al., 1978).
the
normal
is dependent on
To further clarify
which life cycle stages induce filarial hyporesponsiveness,
jirds were infected intraperitoneally with L3 irradiated with
increasing doses of gamma radiation.
MATERIALS AND METHODS
Animals and Experimental design:
Inbred
obtained
female
from
jirds
Tumblebrook
(Meriones
Farms
unguiculatus)
(West
Brookfield,
were
MA) .
Animals were maintained on standard rodent chow and water ad
libitum.
Groups
of
12
to
16
jirds
were
inoculated
intraperitoneally with 100 L3 exposed or not to different
doses of gamma radiation.
An additional group of jirds was
used as an uninfected control.
Necropsies were performed on
3 or 4 jirds per group at 7, 14, 28 and 118 DPI.
Parasites:
L3 were collected from crushed Aedes aegypti mosquitoes
after sedimentation in a Baermann apparatus as previously
73
described (Klei et al., 1990).
Irradiation of L3 was carried
out by exposure of 1 ml syringes containing 100 L3 in 0.5 ml
HEPES buffered RPMI-1640 medium
(GIBCO,
Grand Island,
NY)
supplemented with penicillin (100 units/ml) and streptomycin
(100 /ig/ml) to a 60 Co source at 1447 krads/min.
exposed
to
5
different
increasing
levels
of
L3 were
radiation.
Radiation doses for each group were 0, 15, 25, 35, 45, and 90
krads, respectively.
Antigen:
SAWA used to coat beads was prepared from frozen, mature
adult female and male 23. pahangi
worms by methods previously
described (Klei et al., 1988).
Adult worms were recovered
from female jirds with patent IP B. pahangi infections (>60
DPI) .
Measurement of lung granuloma areas:
Three days prior to necropsy jirds were inoculated in
the
retroorbital
sinus
with
SAWA-
or
diethylamine-(DEA)
coated CNBr-4B beads (Sigma Chemical Co, St. Louis, MO) that
embolized in the lungs as previously described (Klei et al.,
1988) .
Each jird received 2.5 x 104 beads diluted in 0.5 ml
of 0.05% saline.
At necropsy, lungs were perfused with 10%
formalin via the trachea.
Tissues were embedded in paraffin
and step sectioned at 2.5 /*. Several sections from each lung
were cut at 50 /xm intervals and were stained with hematoxylin
and
eosin.
Areas
of
20
granulomas
around
40
to
60
/im
diameter beads were measured in each animal using a image
74
analysis system (Bioquant IV, Biometrics, Nashville, TN ) .
Samples collected:
Animals
were
retroorbital sinus.
bled
prior
to
euthanasia
from
the
MF were counted in 20 fil of fresh blood.
The peritoneal cavity of each jird was washed extensively
with phosphate buffered saline (PBS) . Peritoneal washes were
collected
and cells
were
concentrated by
centrifugation.
Total peritoneal cell numbers were determined with the aid of
an automated counter
Hialeah, Fla).
Isoton
II
(model ZF, Coulter electronics,
Inc,
Peritoneal samples were diluted 1:500 with
coulter balanced electrolyte
solution
(Coulter
Corporation, Miami, FL) . Peritoneal cell differentials were
determined on cytospin preparations. Two cytospins were made
of each sample and stained either with a modified Giemsa
stain or with a non-specific esterase
and Crosby, 1971) .
(NSE) stain
(Yam, Li
Peritoneal MF were counted of 20 /xl of
total concentrated peritoneal fluid and are expressed as the
mean number collected per animal's peritoneal cavity.
were recovered from peritoneal washes.
Worms
Carcasses were soaked
for more than one hour and examined to recover remaining
worms.
Free floating peritoneal granulomas with or without
trapped parasites were collected and fixed in 10% formalin
fixative.
Microscopic pathology of peritoneal granulomas:
After fixation granulomas were dehydrated in a graded
ethanol series to absolute ethanol and were then infiltrated
75
and embedded in Glycol Methacrylate
Polysciencen, Inc. Warrington, PA).
(JB-4 Embedding Kit,
Sections were cut at 2
to 3 jim with a glass knife and mounted on clean glass slide
which
were
heated
to
60°C to
attach
sections.
Routine
hematoxylin and eosin stain was performed on sections.
Parasite measurements and development:
Worm lengths were determined with the aid of a camera
lucida by drawing the outline of the worms.
Lengths of these
drawings
analysis
were
measured
with
an
image
system
(Bioquant IV, Biometrics, Nashville, TN) . Worm lengths were
calculated in mm.
Parasite
development
and
sex
(when
possible)
were
determined following morphological descriptions of B. pahangi
stages previously reported (Schacher, 1962a).
Statistical Analysis:
Statistical comparisons were made between groups using
the
Tukey's
Studentized
Range
Test
for
variability.
Differences were considered significant with P values < 0.05.
RESULTS
Parasite recovery.
Recoveries of parasites irradiated with 25, 35, 45 or 90
krads decreased significantly over time in comparison with
normal parasites
(P < 0.05)
(Table 3.1.).
The trend of
decrease of irradiated parasites was inversely related to the
doses of irradiation.
Parasites irradiated with 45 or 90
krads were not recovered at 118 DPI.
Table 3.1. Parasite recovery from jirds inoculated IP with normal or
irradiated L3 at different times post infection *.
Ri4DIATION DOSES:
0 krads
(4)
15 krads
(3)
25 krads
(3)
35 krads
(3)
45 krads
(3)
90 krads
(3)
7 DPI*
31 ± 21.6
45 ± 18.2
47 ± 18.6
13.3 ± 4.1
17.3 ± 3.9
17 ± 7.4
14 DPI
19 ± 10.7
17 ± 20.5
19 ± 11.34
6.3 ± 1.25
2.33 ± 1.7
1.7 ± 1.2
28 DPI
36 ± 17.7
25 ± 16.3
22.7 ± 21
7 ± 3.6
18 ± 18.8
4 ± 4.32
118 DPI
24 ± 16.7
5.7 ± 3.9
2 ± 1.4
0.33 ± 0.5
0
0
* Results are expressed as mean number of parasite recovery ± standard deviation,
a Days post infection with Brugia pahangi.
() Number of animals in each necropsy time period within a treatment group.
77
Parasite measurements and development.
Worm
lengths
followed
a
trend
similar
to
parasite
recovery and were inversely related to radiation dose (Table
3.2.) . Parasite lengths did not vary significantly among the
different treatment groups at 7 DPI.
However,
lengths of
irradiated parasites at 14, 28 and 118 DPI were significantly
reduced when compared to those of non-irradiated parasites (P
< 0.05).
Gamma radiation at 35, 45 or 90 krads prevented
larval molt to the adult stage.
Some parasites irradiated
with 15 or 25 krads developed into adult female worms and
stunted larvae with features of L 4 .
the
uteri
of
female
worms,
No MF were observed in
which
developed
following
irradiation.
Pulmonary granulomatous response.
The PGRN peaked at 14 DPI in groups inoculated with
normal L3 or L3 irradiated with 15,
0.05)
(Fig. 3.1.).
35 and 45 krads
(P <
Downregulation of the PGRN was observed
in groups that received non-irradiated parasites or parasites
irradiated with 15 krads (P < 0.05) . No significant decrease
of PGRN occurred in groups that received parasites irradiated
with more that 15 krads.
Although a trend of decrease in
PGRN after 14 DPI was observed in the animals that received
parasites irradiated with 25 krads,
statistically
significant.
this decrease was not
Animals
that
received
non­
irradiated parasites had small granuloma areas at 118 DPI
which
did
not
differ
from
those
of
controls
but
were
Table 3.2.
Lengths and development of Brugia pahangi exposed to increasing
doses of irradiation or to no irradiation *.
R A D IA T IO N D O S E S :
0 krads
1.9 ± 0.05
15 krads
25 krads
1.82 ± 0.02 1.81 ± 0.07
35 krads
45 krads
90 krads
1.6 ± 0.05 1.62 ± 0.02 1.73 ± 0.04
7 DPI
L3 *
L3
5.4 ± 0 .36 4.5 ± 0.05
L3
L3
L3
4.17 ± 0.2
3.5 ± 0.22
L4
L4
2.9 ± 0.3
I>3
2.1 ± 0
14 DPI
L4
L4
L4
L4
28 DPI
10.3 ± 0.4
7.6 ± 0.97
Hale
6.9 ± .12
L4, Female
Female, L4
L4
L4
15.7 ± 1.05
Hale
23.3 ± 0.6 18.9 ± 1.25
—
—
12.5 ± 0.5
Female
118 DPI
38.8 ± 2.3
Female, MF
*
a
Female
4.8 ±
0.2
4.6 ± 1.9
2.85 ± 0.2
L4
--
Female
Results are expressed as mean of parasite lengths (mm) ± standard deviation,
Developmental stages found at each time period in the different treatment
groups are indicated.
79
7 DPI
14 DPI
□
E 40
28 DPI
118 DPI
Control
0
15
25
35
RADIATION DOSE, L3
Figure 3.1.
Pulmonary granulomatous response to parasite
antigen in jirds inoculated with normal L3 or with L3 exposed
to increasing doses of gamma radiation.
Mean values with
different letters are significantly different (P < 0.05).
Bars represent standard deviation of the mean.
80
significantly smaller than those of animals that received
parasites irradiated with 15 krads (P < 0.05).
Peritoneal inflammatory response.
MAC accumulation was used as measurement of peritoneal
inflammation.
The MAC was the inflammatory cell type most
abundant
in peritoneal washes of
animals.
Pronounced increase of peritoneal MACs was observed
at
118
DPI
in
the
group
that
infected and uninfected
received
non-irradiated
parasites when compared to any other group (P < 0.005)
3.2.).
However,
received
peritoneal MAC numbers from animals that
irradiated
parasites
were
different from uninfected controls.
peritoneal
cytospins
neutrophils
and
eosinophils
higher
at
infected
in
28
or
eosinophils
(Fig.
included
mast
animals
and
118
control
from
significantly
Other cells found in the
eosinophils,
lymphocytes,
cells.
Numbers
receiving
non-irradiated
DPI
when
groups
jirds
not
(P
receiving
compared
<
of
peritoneal
with
0.05).
irradiated
L3
the
were
other
Peritoneal
L3
did
not
increase consistently throughout infection or among different
treatment groups.
Granulomas partially or totally surrounding irradiated
and non-irradiated parasites were found free floating in the
peritoneal
cavity
granulomas
were
Histologically,
(Fig.
3.3.).
associated
these
with
The
dead
granulomatous
majority
or
of
these
damaged
worms.
lesions
were
characterized by collections of MACs and eosinophils, with
81
60
to
O
X
OT
T
50 40-
■
7 DPI
■
14 DPI
□
28 DPI
H 118 DPI
"5
O
30IU
0)
z
(0
a>
20
-
c
+o
-•
a>
o.
10-
Control
0
15
25
35
RADIATION DOSE, L3
Figure 3.2.
Peritoneal macrophages in jirds inoculated with
normal L3 or L3 exposed to increasing doses of gamma
radiation.
Peritoneal macrophages are represented as non­
specific esterase (NSE) positive cells.
Bars represent
standard deviation of the mean.
82
Figure 3.3
Photograph of a S. pahangi larva surrounded
by a peritoneal granuloma after 14 days post infection.
83
fewer
lymphocytes
and
neutrophils
(Fig.
3.4.).
These
granulomas were sometimes accompanied by occasional foci of
necrosis
and/or
organizing
fibrous
tissue
deposition.
Sections of dead or degenerating worms were found surrounded
by this granulomatous
infiltrate.
Granulomas
inoculated with normal L3 had MF at 118 DPI.
granulomas
appear
to
occur
more
from jirds
At 7 and 14 DPI
frequently
in
jirds
inoculated with irradiated L3 than in those inoculated with
non-irradiated L 3 . However, at 118 DPI granulomas were found
only in jirds inoculated with normal L 3 . No differences in
the
cellular
types
or
histopathological
features
of
granulomas collected from different treatment groups could be
discerned.
MF counts.
MF were present in large numbers (319.5 x 103 ± 38.5 x
103) at
118
DPI
in
all
irradiated parasites.
the
animals
that
received
non­
One out of four animals that received
non-irradiated L3 had MF in peripheral blood at 118 DPI.
No
MF were present in peripheral blood or peritoneal cavity of
animals that received irradiated parasites.
DISCUSSION
Our
experiments
demonstrated
that
gamma
radiation
inhibits the growth and decreases the survival of B. pahangi
as previously reported
al., 1993),
(Oothuman et al.,
1978; Devaney et
These authors also found no MF in female worms
and few or no recognizable male worms.
Similarly, parasite
84
Figure
3.4
Photomicrograph of
the
granulomatous
inflammation induced by B. pahangi larva 14 days post
infection.
Macrophages and eosinophils are numerous and
adhere to the surface of the degenerating L 4 .
85
survival and worm lengths appeared to be inversely related to
the irradiation dose.
Oothuman et al.
(1978) decribed the
effects
and
on
of
10,
25
subcutaneously into cats.
45
krads
L3
inoculated
This report differs with ours in
that 25 kads prevented the worms from developing beyond the
early L 4 .
In our experiment irradiation with 15 or 25 krads
allowed development of some parasites into infertile female
adults.
These slight differences could be related to the
different route of inoculation chosen, to differences in the
responses that the jird and cat may have to B. pahangi or to
variations in calibration of the radiation source.
Downregulation of the PGRN did not occur in animals that
received
L3
irradiated
with
25,
35,
45
and
90
krads.
However, non irradiated parasites or those irradiated with 15
krads induced downmodulation of the granulomatous response.
These findings suggest that viable adult worms are important
in the induction of a hyporesponsive state.
Nevertheless,
these observations do not rule out with certainty that the L4
stage may have some influence.
Furthermore,
the effect of
parasite burden on the PGRN could not be discerned with
clarity.
Small numbers of viable female worms were found in
animals that received L3 irradiated with 25 krads, but the
PGRN was
not
significantly decreased.
In addition,
the
degree of PGRN downmodulation was greater in the animals that
received
normal
L3
than
irradiated with 15 krads.
in
animals
that
received
L3
This difference in the degree of
86
granulomatous downmodulation may be related to the presence
of
MP
and/or
the
requirement
of
active
adult
worms
not
damaged by irradiation, or to higher total parasite burden.
Results
in
Chapter
2
demonstrated
that
parasite
burden
effects the PGRN, since high numbers of female worms induced
smaller granuloma
Brosshardt et al.
areas
than lower numbers.
Studies
by
(1995) showed that the degree of antigen-
mediated granulomatous downregulation and the decrease in
splenocyte
proliferation
circulating
parasite
MF
and
burden
correlated
adult
female
influences
with
worms
positively
the
number
indicating
the
of
that
degree
of
hyporesponsiveness.
It
is
very
likely
that
MF
contribute
to
filarial
hyporesponsiveness as suggested by others (Klei et al., 1990;
Brosshardt et al., 1995).
Results in chapter 2 demonstrated
that inoculation of MF alone induces downregulation of the
PGRN.
Prolonged IP exposure to MF or repeated
single) .subcutaneous
inoculations
of
MF
antigen
(but not
in
mice
result in a Th2 response (Pearlman et al., 1993a; Pearlman et
al., 1993b), thereby indicating that chronicity and parasite
burden may play a decisive role.
The lack of PGRN downmodulation in animals that received
L3 exposed to high doses of irradiation may also be explained
by
the
impaired release of
immunomodulation.
inhibits
protein
certain
antigens
relevant
in
It has been demonstrated that irradiation
synthesis
in
Schistosoma
mansoni
87
schistosomula
(review in Wales and Kusel,
1992).
Recent
studies in B. pahangi did not reveal consistent differences
in protein synthesis between irradiated and non-irradiated L3
or L4 (Devaney et al., 1993).
not studied.
support
However, the adult stage was
The present results and data in chapter 2
the
importance
of
adult
stages
for
inducing
downmodulation in the absence of MF as other experiments
corroborate (Brosshardt et al., 1995; Lin et al., 1995).
The profile of cytokine mRNA in jirds subcutaneously
infected with B. pahangi follows a Th2 pattern that coincides
with
the
hyporesponsiveness
state
chronic period in this model
characteristic
(Mai,
1996)
of
the
and mimics
the
cytokine response reported in microfilaremic asymptomatic
human patients (King et al., 1993; Maizels et al., 1995) . It
has been proposed that the downregulation of Thl cytokines in
filariasis
diminishes
pathology
and
cellular
clearance
of
effectors
parasites
that
lead
(Nutman,
to
1995).
Vaccination studies in the jird with radiation attenuated-B.
pahangi
L3
increased
showed
severity
(Petit et al.,
that
of
1993).
resistance
lymphatic
is
lesions
associated
with
and
PGRN
higher
It is possible that the high PGRN
associated with protection
is mediated by
a ThO
or
Thl
phenotype.
Peritoneal inflammatory response,
as measured by MAC
numbers, was most pronounced at the chronic time period in
jirds
receiving
the
non-irradiated
L3.
This
prominent
88
inflammatory response was associated with the presence of
high numbers of intraperitoneal MF, indicating that MF may
act
in
certain
stimulus.
circumstances
as
a
potent
inflammatory
MF has been described in granulomatous lesions in
the jird (Vincent et al., 1980; Klei et al., 1982; Jeffers et
al., 1987), ferret (Crandall et a l ., 1982) and humans (Meyers
et al., 1976).
Free floating peritoneal granulomas in jirds
inoculated with normal L3 were found more frequently at the
time when MF were present.
circulating
in
microvasculature
apparent
MF
are most
commonly
peripheral
blood
and
of
organs
without
diverse
inflammatory
response.
found
throughout
the
inducing
any
Compartimentalization
phenomena driven by localized immune responses yet to be
defined may result in entrapment and killing of MF followed
by granulomatous inflammation.
In our study, the peritoneal
inflammatory
not
response
downregulated
PGRN
did
suggesting
correlate
that
peritoneal
inflammation
exist
responsible
for
responses.
systemic
with
factors
independently
Such
the
inducing
of
those
difference
in
responses suggests that diverse body compartments differ in
the ability to cope with the parasite, which may be related
to
the
nature
interactions
as
of
parasite-
well
as
and/or parasite
particular
recruited to such body locations.
cell
product-cell
types
that
are
This may also explain the
differences in the kinetics of proliferative responses to
SAWA of spleen,
axillary lymph node and renal lymph node
89
cells
from
jirds
adult parasites
lymph
nodes
inoculated subcutaneously and harboring
in lymphatic vessels that drain to renal
(Klei
et
al.,
1990;
Rao
Variability in regional and systemic
et
al.,
immune
1995).
responses
to
parasite antigen have also been found in the dog infected
with
B.
pahangi
(Miller
et
al.,
1991)
as
infections with gastrointestinal nematodes
well
as
in
(Kelly et al.,
1991).
The
peritoneal
cavity
appears
to
be
an
appropriate
compartment to study parasite cell-interactions and may be
very
advantageous
to
characterize
further
immunological
properties of cells such as MACs which participate in the
immune and inflammatory response to filaria.
recruited in large numbers
response
component
However,
to
B.
of
scant
pahangi
into the peritoneal
infection
granulomatous
information
The MAC is
and
lymphatic
has
been
is
also
lesions
further considered in chapter 4.
a
central
and
collected
significance of the MAC in filarial infections.
cavity in
PGRN.
about
the
This will be
CHAPTER 4.
MACROPHAGE ACTIVATION AND TNF PRODUCTION IN
JIRDS INFECTED WITH FEMALE OR MALE BRUGIA
PAHANGI
INTRODUCTION
Wuchereria bancrofti and Brngia malayi are lymphaticdwelling filarial nematodes that infect humans in tropical
and subtropical regions of the world.
The pathology caused
by
characterized
filarial
granulomatous
parasites
is
inflammation
primarily
to
the
parasite
and
by
a
parasite
products that has been attributed to a state of specific
filarial
Ottesen,
immune
1980).
hyperresponsiveness
(Lichtenberg,
1957;
Lymphatic dilatation in absence of marked
inflammation has been reported in microfilaremic asymptomatic
patients (Jungmann et al., 1991) and has been reproduced in
immunodeficient mice (nu/nu and SCID)
Nelson et al., 1991).
(Vincent et al., 1984;
Lymphatic changes in absence of marked
inflammation have been interpreted as an example of specific
immunosuppression and are probably related to the persistent
mechanical action of adult worms within lymphatic vessels
(Von Lichtenberg, 1987).
Immune reconstitution in nude mice
with T cells from filarial sensitized immunocompetent mice
results in development of a granulomatous reaction within and
around lymphatics proving the importance of T lymphocytes in
disease manifestations (Vickery et al., 1991).
are
typically
associated
with
Intact worms
lymphangiectasia,
whereas
damaged or dead worms are always accompanied by inflammation
and
lymphatic
obstruction
(Rifkin
Wartman, 1947; Lichtenberg, 1957).
90
and
Thompson,
1945;
91
Immunological
studies
in microfilaremic
asymptomatic
human patients have revealed depressed lymphoproliferative
responses
(Ottesen et
al.,
1977;
Piessens
et
al.,
1980;
Nutman et al., 1987a; King et al., 1992) to filarial antigen
and production of Th2 cytokines
(IL-4 and IL-10)
al., 1993; Maizels et al., 1995).
lymphatic
enhanced
pathology
blastogenic
are
Conversely, subjects with
usually
responses
amicrofilaremic,
to
filarial
produce Thl cytokines (IFN-y and IL-2).
potent
negative
(King et
have
antigens
and
IL-4 and IL-10 have
immunoregulatory functions
by which
they
suppress cell-mediated immune responses and favor persistence
of the parasites (Hart et al., 1989; Martinez et al., 1990;
Bodgan et al.,
1991;
De Waal Malefyt et al.,
1991).
It
appears obvious that the deviation of the immune response
towards
a Th2 pattern controls
inflammatory responses
in
microfilaremic asymptomatic patients (Nutman, 1995).
In the jird
pahangi,
the
development
implication
of
demonstrated.
(Merionea unguiculatus)
of
lymphatic
the
immune
granulomas
Presensitization
and
infected with B.
response
has
in
been
protective
the
well
immunity
results in increased severity of lymphatic lesions (Klei et
al., 1982; Petit et al., 1993).
Chronic microfilaremic jirds
manifest
state
a
filarial
specific
of hyporesponsiveness
(Lammie and Katz, 1983) accompanied by decreased lymphatic
lesion severity (Klei et al., 1981; Klei et al., 1990).
92
Measurement of cytokine mRNA from spleen and lymph nodes of
B. pahangi-infected jirds revealed IL-4 and IL-10 messages as
early as 28 days post infection (DPI), followed by a marked
increase of these cytokines at 56 and 150 DPI
This
pattern
of
cytokines
mimics
that
(Mai, 1996).
described
in
hyporesponsive filariae-infected human patients (King et al.,
1993; Maizels et al., 1995).
The MAC is the main cellular component of the granuloma
and is often present covering the surface of filariae (Nelson
et al., 1976; Jeffers et al., 1987),
(chapter 3) and other
nematodes (Soulsby, 1963; Jeska, 1969).
is
a
major
component
multicellular
in
parasites,
the
inflammatory
its
role
immunomodulation during helminth
studied extensively.
Even though the MAC
in
reactions
pathology
infections
has
not
to
and
been
The implication of T cell-mediated MAC
activation in the destruction of metazoan parasites has only
been amply investigated in schistosomiasis, where it has been
shown that MAC activation is important in resistance (Civil
et al.,
James
1978; Mahmoud et al.,
et
al.,
1982b).
1979;
The
James et al.,
prominent
1982a;
granulomatous
inflammation around schistosoma eggs characteristic of the
acute state of the disease is gradually downmodulated as the
infection progresses to the chronic phase.
MAC
in
schistosoma
"immunomodulation"
has
The role of the
been
reviewed
(Stadecker, 1994) . MACs from egg granulomas induce anergy in
Thl lymphocytes (Stadecker, 1992; Stadecker et al., 1990) and
93
in Th-1 clones specific for egg antigens (Flores Villanueva
et
al.,
1994).
downmodulating
Granuloma
MACs
the granulomatous
Villanueva et al., 1994).
are
also
lesions
in
capable
vivo
of
(Flores
Further, IL-10 was found to exert
a potent downregulatory effect on antigen presenting cells.
Culture of granuloma MACs in the presence of anti-IL-10 mAb
resulted in upregulation of MHC class II molecules, and the
costimulatory molecules B7-1 and B7-2 on granuloma MACs as
well as in the ability to stimulate egg antigen-specific T
cell responses (Stadecker, 1994).
Rat
and mouse
s
have
been
shown
to participate
in
cytotoxic reactions presumably mediated by antibody and/or
complement
against
Brugia
spp MF
(Oxenham et
al.,
1984;
Chandrashekar et al., 1985; Chandrashekar et al., 1986) and
L3
(Chandrashekar et al.,
1985)
in vitro.
Studies in the
jird have demonstrated that B. pahangi infection results in
increased
capacity
of
MACs
to
phagocytize
and
kill
Staphylococcus aureus (Jeffers et al., 1984).
The purpose of the experiments described in this chapter
was to determine the level of jird MAC activation and TNF
production following in vivo infection with B. pahangi.
The
effect of different parasite stages (female or male worms) on
MAC function was also investigated.
94
MATERIAL AMD METHODS:
Animals
Inbred,
female,
unguiculatus) were
Brookfield,
MA) .
6
to
obtained
Inbred,
8
week
old
jirds
from Tumblebrook
female,
(Meriones
Farms
(West
Balb/c mice were kindly
provided by Dr. J.L. Krahenbuhl and were originally obtained
from Jackson Laboratories (Bar Harbor, Maine). Animals were
maintained on standard rodent chow and water ad libitum.
All
jirds and mice used in these experiments were infected at 3
to 4 months of age.
Jn vivo infection with BCG
Briefly, one group of 24 jirds and one group of 24 mice
were
inoculated
intradermally
(ID)
with
3 x
10s colony-
forming units of living Mycobacterium bovis, strain bacillus
Calmette-Guerin
(BCG)
(kindly
provided
by
Dr.
J.L.
Krahenbuhl) . 24 and 48 hours before euthanasia BCG immunized
animals were inoculated intraperitoneally (IP) with 50 ug of
purified protein derivative of the tubercle bacillus
(PPD)
(Parke-Davis, Rochester, MI) dissolved in 0.5 ml of phosphate
buffered saline (PBS) . The BCG-PPD immunization protocol has
previously been described (Ruco and Meltzer, 1977) . Control
animals, 24 per group, were inoculated ID with 0.05 ml of PBS
and IP with 0.5 ml of PBS.
Necropsies were performed at 15,
28 and 42 days post infection (DPI) with BCG.
Brugia Parasites
The
aegypti
B.
and
pahangi
jirds
life
cycle
was
maintained
as previously described
(Klei
in
et
Aedes
al,,
95
1990).
Male and female adult B. pahangi were aseptically
collected from the peritoneal cavities of jirds with chronic
patent IP infections.
The worms were washed in RPMI-1640
medium (GIBCO, Grand Island, NY) supplemented with penicillin
(100
U/ml)
and
streptomycin
transfered to 3 ml syringes.
(100
/xg/ml)
prior
to
being
Single sex implantations of 10
female or 10 male worms into the peritoneal cavity of jirds
were done using 16 gauge needles.
inoculated IP with RPMI medium.
Control
animals were
Necropsies were performed at
15, 50 to 56, and 135 DPI.
Macrophage culture
Peritoneal cells were aseptically collected from the
peritoneal cavities of jirds and mice in PBS containing 10
units/ml of heparin (Sigma).
once
at
250 x g
Peritoneal cells were washed
for 10 min and transfered
to RPMI-1640
supplemented with antibiotics (described above) , HEPES buffer
(25 mM) , 2 x 10'5 M 2-mercaptoethanol, L-glutamine (2 mM) and
10% heat inactivated fetal bovine serum
prepared on LUX cover slips
(FBS).
Cells were
(Miles Scientific,
Div. Miles
Laboratories, Inc., Naperville, IL) in 24-well tissue culture
plates
(GIBCO, Grand Island, NY) at 3 x 106 cells/well and
were incubated at 37°C.
adherent
After 4 to 6 hours of culture non
cells were washed off by lavage with PBS.
MAC
monolayers on the coverslips were used in the bioassays for
Toxoplasma killing and for tumor necrosis factor (TNF) and
Nitric oxide (NO) production.
96
Spleen cell culture and collection of macrophage activating
factors
Macrophage activating factors (MAF) were collected from
cultures of spleen cells from BCG-PPD inoculated animals or
from untreated animals.
Single cell suspensions were obtained from spleens of
BCG-PPD
inoculated
previously
(Klei
and
et
untreated
animals
as
1990).
Briefly,
spleens
al.,
described
were
removed, placed on a nylon mesh screen and partially minced
with scissors.
Using a 10 cc disposable syringe plunger, the
spleen pieces were pressed thru the screen into a petri dish
containing complete medium.
The spleen cell suspension was
concentrated by centrifugation.
Red blood cells were removed
by suspending the cell pellet in prewarmed NH4C1 solution
(Sigma)
(approximately 3 ml per spleen) and gently shaking
the suspension for 3 min in a 37°C water bath.
Cells were
washed once in complete medium (250 x g, 10 min) and cultured
in 5% FBS, RPMI-1640 medium supplemented as indicated above.
To obtain BCG-PPD MAF, spleen cells were incubated at a
concentration of 4 x 106 cells/ml with 40 (ig/ml of PPD in 25
cm2 plastic tissue culture flasks
(Costar,
Cambridge,
(Nacy
and
hours,
et
al.,
1985).
After
48
72
MA)
culture
supernatants were collected, centrifuged at 10,000 x g for 10
min and stored at -20°C until use.
To obtain Con A MAF, spleen cells were incubated at a
concentration of 1.5 x 106 cells/ml with 1, 2 or 3 /ig/ml of
97
Con A (Sigma) in 25 cm2 plastic tissue culture flasks for 24,
48,
72,
96
or
120
hours.
Culture
supernatants
were
collected, centrifuged at 10,000 x g for 10 min and stored at
-20°C until use.
Previous work in our laboratory and by
other investigators (Lammie and Katz, 1983) have demonstrated
that the concentrations of Con A used at the different time
intervals to obtain MAF induced spleen cell proliferation
measured by tritiated Thymidine incorporation.
Reagents
Bacterial lipopolysaccharide (LPS) from Escherichia coli
0111.B4
was
used
(Sigma).
Recombinant
murine
IFN-y was
originally obtained from Genentech, Inc (South San Francisco,
CA) .
In vitro activation of macrophages
A combination of either BCG-PPD MAF, Con A MAF (1:1, 1:2
or 1:3 MAF/medium) or murine rIFN-y (500 or 1000 U/ml) with
10 or 50 ng/ml of LPS were added to the MAC monolayers.
were cultured undisturbed over night.
MACs
Attempts to activate
jird and mouse MACs in vitro were repeated at least three
times for each treatment.
Toxoplasma killing assay
Tachyzoites
of
Toxoplasma
gondii
RH
strain
were
harvested from the peritoneal cavities of Balb/c mice 2 days
after
infection and purified by
filtration
through
3 fim
polycarbonate membranes (Nucleopore Corporation, Pleasanton,
CA) as described previously (Wilson et al., 1980/ Sibley and
98
Krahenbuhl,
1988).
MAC monolayers from controls and from
animals infected with BCG-PPD or B. pahangi were challenged
with 1.5 x 106 freshly harvested T. gondii cells.
1 hour
later extracellular T. gondii cells were rinsed off and MAC
monolayers were returned to culture.
was assesed after 20 h of culture.
stained
with
Hema
Houston, TX) .
3
(Curtin
Microbicidal activity
Coverslips were fixed and
Matheson
Scientific,
Inc,
The number of intracellular Toxoplasma were
counted in 100 MACs per coverslip.
Triplicate samples were
done for each treatment.
Tumor Necrosis Factor production
Levels
of
TNF-a-like
activity
were
determined
in
supernatants of MAC cultures by a modified L929 fibroblast
cell
lytic assay
1991) .
MACs
(Agarrwal et al.,
1985;
Sibley et al.,
MAC monolayers were prepared as described above.
were
either
unstimulated.
stimulated
with
50
/ig/ml
of
LPS
or
Supernatants were collected after 4 hours,
centrifuged at 10,000 x g for 10 mn and stored at -70°C until
use.
96
Duplicate samples were serially diluted three-fold in
well,
Cambridge,
flat
MA) .
bottoned
tissue
L929 cells
culture
plates
(Costar,
(kindly provided by Dr.
J.L.
Krahenbuhl) were harvested in active, log-phase growth.
L929
cell suspension containing 2 /xg/ml of actinomycin D (Sigma)
was prepared at a concentration of 3 x 105 cells/ml.
100
/il/well of cell suspension was added to each well and plates
were incubated at 37°C,
After 24 hours medium was emptied
99
from plates and 0.1% of crystal violet was added.
were incubated at room temperature for 10 min.
Plates
Plates were
rinsed with water after emptying the crystal violet (Sigma).
100 fil of 1% sodium dodecyl sulfate (SDS)
in each well.
(Sigma) were added
Absorbance was read at 570 nm.
Concentrations
of TNF were calcultated in units defined as the reciprocal
dilution which yields 50% lysis of L929 cells.
Measurement of Nitrite production
Levels
determined
of
nitrite
(N02‘)
in
spectrophotometrically
MAC
at
supernatants
540
nm
were
following
reaction with the Griess reagent (Adams et al., 1990; Drapier
and
Hibbs,
1988) .
N02' is
the
stable
nonenzymatic degradation of nitric oxide
end
(NO).
product
of
N02‘ levels
were measured in supernatants from MAC monolayers treated in
vitro with either IFN-y or Con A MAF and LPS as second signal
or only with LPS.
cultured
MAC
from
N02' production was also determined in
BCG-PPD
immunized
animals.
N02'
concentration was calculated from a NaN02 standard curve and
results are expressed in /iM (micromoles) .
Statistical Analysis
Results were analyised statistically with a comparative
analysis of variance using the Tukey's Studentized Range
test.
100
RESULTS
Macrophage activation a£ter in vivo infection of jirds and
mice with BCG.
Prior to experimental filarial infection, the activation
of
jird MACs
methods.
was
characterized by
in vitro and
in
vivo
All attempts to activate jird MACs in vitro with
supernatants from Con A stimulated spleen cultures or with
murine rlFN-y as a first signal, followed by LPS as second
signal failed (Table 4.1.).
Hpwever, parallel experiments
with murine MACs treated with mouse Con A MAF or murine rlFNy
consistently
Furthermore,
yielded
mouse
Con
positive
A
MAF
or
results
murine
restrict Toxoplasma growth in jird MACs.
however,
(Table
rlFN-y
4.1.).
did
not
Jird Con A MAF,
conferred toxoplasmacidal activity to mouse MACs.
This activity was significantly less than that induced by
INF-y or mouse MAF (P < 0.05).
Alternatively, we chose to activate MACs in vivo with
BCG, as previously demonstrated in the murine system
and Meltzer, 1977).
(Ruco
Results showed that MACs recovered from
BCG-PPD treated mice and jirds restricted the intracellular
growth of Toxoplasma cells when compared to control MACs at
the three time periods tested (15, 28, 42 DPI)
(P < 0.05).
Microbicidal activity of BCG-PPD treated peritoneal MACs was
manifested by a decrease in the percentage of Toxoplasmainfected MACs (Fig. 4.1. and 4.4.), decrease in the number of
Toxoplasma cells per infected MAC (Fig. 4.2. and 4.5.)
and
Table 4.1.
Effects of Con A MAF and murine rlFN-y on the toxoplasmacidal activity of
jird and mouse macrophages*. LPS was added as a second signal.
JIRD MACROPHAGES
Control0
7.13
Murine rlFN-y
+ LPS
Jird Con A MAF
+ LPS
Mouse Con A
MAF + LPS
17.33 ± 0.94
22.67 ± 4.03
21.5 ± 1.5
% INFECTED
MACROPHAGES
22.7 ±
# TOXOLASMA/
INF MACS
3.18
3.35
3.31
3.97
# TOXOPLASMA/
100 MACS
72 ± 22.6
58 ± 10.98
75 ± 12.35
85.5 ± 15.5
MOUSE MACROPHAGES
*
a
c
Control0
Murine rIFN-y
+ LPS
% INFECTED
MACROPHAGES
15.57 ± 0.94
3.67 ± 2.05*
9 ± 1*
4 ± 0.82*
# TOXOPLASMA/
INF MACS
6.64
1.27*
4.22*
1.15*
# TOXOPLASMA/
100 MACS
104 ± 15.77
4.67 ± 2.86*
38 ± 5*
4.6 ± 0.94*
Jird Con A MAF
+ LPS
Results are expressed as mean ± standard deviation,
Indicates a significant difference (P < 0.05) from the controls,
Peritoneal macrophages from control uninfected animals that had no
treatment.
Mouse Con A
MAF + LPS
in vitro
102
JIRD BCG-PPD EXPERIMENT
CONTROL
•.v.v.v
.v.v.v.
co
w
'.V .V .V
'.V .V .V
V.V.V.'
I
V .V .V .'
'.V .V .V
o
as
u
’.V .V .V
**••
v.v.v.
'.V .V .V
.vXv>.
v.v.
.v.v.v.
v.v.v.
V
'.V.V
m
m
i !•
m
w &
'W:
*
wXvXv
•Xv.v.
r.v.v.v
DPI
Figure 4.1.
Effect of BCG-PPD immunization on jird
macrophage
activation measurjed by
the
percentage
of
Toxoplasma-infected macrophages. Days postinfection indicate
days after ID BCG inoculation.
One and two days before
collection of peritoneal macrophages animals were boosted IP
with PPD.
Macrophages from BCG-PPD immunized jirds were
compared with control uninfected jirds. Bars represent the
standard deviation of the mean.,
103
JIRD BCG-PPD EXPERIMENT
E3 CONTROL
■
BCG
...
v.v.v.
•v.v.
4-
:•:•>:•:•:•:
•I*:*:*
•.V.
w.w.%
• ••••
v.v.v.
3-
v.v.v.
.WAV
2- 1 1
ms
m s
*
s m
i f
mv.v.;.;
.
••••••:
•••V.V.
V
0*
:Wx*
••••v.v
■
■
■
•:••:*:•:
■
a*.
DPI
Figure 4.2.
Effect of BCG-PPD immunization on jird
macrophage activation measured by the mean number of
Toxoplasma per infected macrophage. Number of Toxoplasma per
infected macrophage was calculated dividing total number of
organisms in 1 0 0 macrophages by the percentage of infected
macrophages.
104
JIRD BCG-PPD EXPERIMENT
150
# TOXOPLASMA
/ 100 MACROPHAGES
CONTROL
t
BCG
%%%%%%•
100
.•v•.
..
vv.
•v
.v
.%%%%%%
%%%%%%
.%%%%%%
.V•.v.v.
v.v.%
.v.v.v.
50
.v.v.v.
'.V .V .V
v.v.v
.V.V..
vv
.v.v.v
,\vX*%
n
.%%%%%
■
! «
::::::
•%
V.V.%
Figure 4.3.
Effect of B C G r P P D
immunization on jird
macrophage activation measured by the mean number of
Toxoplasma per 100 macrophages.
105
MOUSE BCG-PPD EXPERIMENT
m
OONIROL
BOO
IFN-g+ LPS
•.v.v
.'.V .
•.v.v
•v.v
.v.v
V
'.V .V
,v.v.
v.v.
V
•.v.v
.v.v
V.'.V
v.v.
m
T
DPI
Figure 4.4.
Effect of BCG-PPD immunization on mouse
macrophage
activation measured by
the
percentage
of
Toxoplasma-infected macrophages. Days postinfection indicate
days after BCG inoculation. Effect of in vitro murine rIFN -7
and LPS treatment on macrophage activation is also shown.
Bars represent the standard deviation of the mean.
106
MOUSE BCG-PPD EXPERIMENT
CONTROL
BCG
IFN-g + LPS
# TOXOPLASMA
/ INF MACROPHAGE
10 .
D PI
Figure 4.5.
Effect of BCG-PPD immunization on mouse
macrophage activation measured by the mean number of
Toxoplasma per infected macrophage.
107
MOUSE BCG-PPD EXPERIMENT
/ 100 MACROPHAGES
250
CONTROL
BCG
200-
IFN-g + LPS
T
# TOXOPLASMA
V .V .
.v.v
m
•v.v.
ti
v.v.*
K
II
•.v.v
I
m
T
42
D PI
Figure 4.6.
Effect of BCG-PPD immunization on mouse
macrophage activation measureid by the mean number of
Toxoplasma per 100 macrophages.
108
decrease in the number of Toxoplasma per 100 MACs (Fig. 4.3.
and
4.6.).
Toxoplasmacidal
activity
did
not
vary
significantly in mice or jirds throughout the infection.
Jird MACs
showed a lower capacity to restrict
Toxoplasma
growth than mouse MACs.
Effect of
activation.
BCG-PPD
MAF on murine
and
jird
macrophage
The addition of murine BCG-PPD MAF to MAC monolayers
from control mice resulted in marked increase of microbicidal
activity
measured
via
Toxoplasma
killing
(Table
4.2.).
However,
jird BCG-PPD MAF did not restrict the growth of
Toxoplasma in MACs from control jirds.
Nitrite production
N02' release was not detected in any of supernatants from
treated or non-treated jird MAC cultures treated with Con A
MAF
or
IFN-y plus
LPS or LPS
alone.
However,
parallel
experiments using murine MACs resulted in N02‘ production as
previously
described
(Ding,
1988).
observed in murine
The
production
was
combination
of IFN-y and LPS (P < 0.05).
production occurred over time
(Table 4.3.).
MACs
greatest
treated
N02'
with
a
Increase of N02'
in LPS-treated murine MACs
NO production above control levels was not
found in MACs from BCG-PPD immunized jirds (Table 4.4.) .
Macrophage activation after
intraperitoneal
jirds with adult female or male B. pahangi.
infection
of
Toxoplasmacidal activity was measured in peritoneal MACs
from jirds with intraperitoneal infections with either adult
Table 4.2.
Effects of BCG-PPD MAF on the Toxoplasmacidal activity of jird and mouse
macrophages*. LPS was added as a second signal. BCG-PPD MAF from animals with 15, 28
and 42 days of BCG infection were used.
JIRD MACROPHAGES
Control*
% INFECTED
MACROPHAGES
28.3 ± 0.47
15 DPI BCG
MAF + LPS*
21
±
0.82
Control1*
15.7 ± 1.25
28 DPI BCG
MAF + LPSb
42 DPI BCG
MAF + LPSb
15.5 ± 0.5
12.7 ± 1.69
# TOXOPLASMA/
INF MACs
4.91
4.03
4.29
4.45
3.87
# TOXOPLASMA/
100 MACS
139 ± 5.43
84.7 ± 14
67.3 ± 3.4
69 ± 7
49 ± 8.64
MOUSE MACROPHAGES
Control*
% INFECTED
MACROPHAGES
*
a,b
c
31.3 ± 2.49
# TOXOPLASMA/
INF MACs
6.5
# TOXOPLASMA/
100 MACS
204
± 39
15 DPI BCG
MAF + LPS*
Control1*
28 DPI BCG
MAF + LPSb
7 ± 5e
13.3 ± 1.7
1
1.28°
8.5
lc
9 ± 7C
146.7 ± 18
1
± 0C
IFN-V +
LPSb
1
± 0C
lc
± 0°
Results are expressed as mean ± standard deviation.
Treatment groups with the same letter were done at the same time.
Indicates a significant difference (P < 0.05) from the controls.
1
± 0C
110
Table 4.3.
Nitric oxide production by murine and jird
macrophages*. Results are expressed as fiM of N02*. Note the
absence of N02* release from jird macrophages.
Maximum N02‘
production is present in murine macrophages treated with IFNy and LPS.
HOURS OF
CULTURE
JIRD MACROPHAGES
MOUSE MACROPHAGES
No treatment
4 hours
0
0
24 hours
0
0
LPS treatment
4 hours
0
1.49 ± 2.11
hours
0
12.96 ± 0.57*
24 hours
0
39.92 ± 2.25*
12
IFN-y + LPS
20
hours
52.46 ± 1.31*
0
Jird Con A MAF + LPS
20
hours
ND
0
Medium
0
*
a
ND
hours
0
Results are expressed as mean ± standard deviation,
Indicates a significant difference (P < 0.05) from
samples with no treatment.
Not determined.
Table 4.4. Nitric oxide production by macrophages from jirds and mice immunized
with BCG-PPD*. Results are expressed as fiH of N02‘.
DAYS POST
INFECTION*
TX
15 DPI*
0
ITX
CONTROL0
4h
LPS
0
0
12h
4h
LPS
0
12h
LPS
42 DPI
0
4h
LPS
0
LPS
*
a
b
12h
BCG-PPD5*
2
± 0.8
JIRD MACROPHAGES
CONTROL0
2
± 0. 8 8
BCG-PPD5*
0.65 ± 0.7
2.4 ± 0.3
1.7 ± 0.5
0.8 ± 0.5
13.4 ± 14
1.3 ± 0
1.12
5.5 ± 0.8
22.7 ± 6.6
0
0.9 ± 1.28
0.15 ± 0.22
1.16 ± 0.7
1.4 ± 0.6
1.4 ± 0.76
ND
3.33 ± 1.4
0.8 ± 0.5
1.46 ± 0.42
ND
0.17 ± 0.19
0
0.4 ± 0.44
ND
14.6 ± 4.4
0.3 ± 0.29
0.29 ± 0.38
0.32 ± 0.45
4.6 ± 4.3
0.4 ± 0.5
0.54 ± 0.94
0.63 ± 0.6
4.7 ± 6.7
0.17 ± 0.3
0.8 ± 0.89
ND
14.3 ± 13.9
0
0
ND
34.8 ± 20.8
0
0
ND
± 1.2
Results are expressed as mean ± standarddeviation,
Number of days post infection
(DPI) with BCG.
Animals were infected with BCG and shortly before euthanasia they were
boosted IP with PPD.
Uninfected animals.
Macrophages were cultured with LPS or with no LPS.
Indicates hours of culture with LPS or with no LPS.
Not determined
111
c
TX
ITX
ND
1.29 ± 0.51
1.5 ± 0.39
LPS
28 DPI
MOUSE MAC ROPHAGES
112
female or male B. pahangi, and was compared to that of MACs
from uninfected jirds or from BCG-PPD immunized jirds.
MACs
from jirds infected with adult female or male B. pahangi at
15 DPI restricted the growth of Toxoplasma cells in a manner
similar
to
Percentage
that
of
of
MACs
infected
from
MACs,
BCG-PPD
number
immunized
of
jirds.
Toxoplasma
per
infected MAC and total number of Toxoplasma per 100 MACs at
15 DPI were decreased with respect to control MACs (P < 0.05)
(Fig. 4.7., 4.8. and 4.9.).
MACs from female or male worm
infections at 50 or 135 DPI showed percentage of infected
MACs, number of mean Toxoplasma per infected MAC and total
number of Toxoplasma per 100 MACs significantly higher than
MACs
from
BCG-PPD
immunized
jirds
different from uninfected controls
and
not
significantly
(P < 0.05)
(Fig. 4.7.,
4.8. and 4.9.).
TNF-like production by peritoneal macrophages
infected with adult female or male B. pahangi.
Spontaneous
release
erratically in MACs
jirds
throughout
stimulated
MACs
of
TNF
controls
occurred
from female and male worm-inoculated
infection.
was
above
from jirds
increased
Release
with
of
TNF
respect
from
LPS-
uninfected
controls at 15, 56 and 135 DPI in the female worm infection,
and at 15 and 56 DPI in the male worm infection (Table 4.5.) .
In both female and male worm infections the LPS-stimulated
TNF production peaked at 56 DPI and decreased markedly at 135
DPI .
113
BRU GIA PAH ANG I
EXPERIMENT
CONTROL
FEMALE
co
w
%
S3
o.
□
MALE
BCG
o
u
«<
s
Q
W
H
U
to.
v.v
V.'.V
...V .
.v.v.
■MM.
D PI
Figure 4.7.
Macrophage activation in jirds inoculated IP
with female or male B. pahangi measured by the percentage of
Toxoplasma-infected
macrophages.
The
toxoplasmacidal
activity of macrophages from jirds infected with female or
male worms at 15 DPI was similar to that of macrophages from
BCG-PPD immunized jirds. Bars represent standard deviation
of the mean.
114
BRUGIA PAHANGI
EXPERIMENT
E3 CONTROL
FEMALE
W
O
.......
□
£
MALE
BCG
g
•v.v*
u
<
S
•iV.W
Q
.v.v
VA
S
U
*,VA
AVA
•A 'A
<
S
CO
<5
J
cu
o
X
O
H
%
W
A !A
AVA
v:¥:
AVA
A'A*
A \V
•A 'A
>Xv.
AVA
D PI
Figure 4.8.
Macrophage activation in jirds inoculated IP
with female or male B. pahangi measured by the number of
Toxoplasma per infected macrophage. Toxoplasmacidal activity
of macrophages from uninfected or BCG-PPD infected jirds is
also shown.
115
BRUGIA P AH AN G I
EXPERIMENT
CONTROL
FEMALE
CO
Ed
□
MALE
S
■
BCG
S3
cu
O
OSS
u
<
V .'.Y
W .V
2
fi
Ed
H
O
Ed
V .V
£
.v.v.
.v.v.
v.v.
.v.v.
D PI
Figure 4.9.
Macrophage activation in jirds inoculated IP
with female or male B. pahangi measured by the total number
of Toxoplasma per 100 macrophages.
Bars represent standard
deviation of the mean.
Toxoplasmacidal
activity of
uninfected or BCG-PPD infected jirds is also shown.
Table 4.5.
Tumor necrosis factor-like production by peritoneal macrophages from jirds with
female or male B.pahangi* . Macrophage monolayers were nontreated (no LPS) or stimulated with
50 ng of LPS for 4 hours.
TNF production was quantitated with the L929 cell line and
expressed as units/ml. Note the marked increase of LPS-induced TNF that occurred in female
and male worm, infections at 15 and 56 DPI when compared with controls.
Chronic worm
infection resulted in prominent downregulation of TNF release.
15 DPI
56 DPI
135 DPI
No LPS
LPS
No LPS
LPS
No LPS
LPS
FEMALE WORM
INFECTION
12.3 ± .2
95 ± 73.8
0.9 ± 1.4
173 ± 83
7.7 ± 4.6
42 ± 9.5
MALE WORM
INFECTION
3.6 ± 0.8
24 ± 2.5
3.2 ± 0.6
138 ± 57
2.3 ± 2.3
5.5 ± 2.3
CONTROL6
2.4 ± .04
8.7 ± 0
1.2 ± 0.9
7.14 ± 4
3 ± 0.18
*
c
DPI
6
± 3.7
Results are expressed as mean ± standard deviation.
Uninfected controls
Days post infection with B. pahangi
H
<n
117
DISCUSSION
Most MAC activation systems consist of IFN -7 as a first
signal
and
a
second
signal
represented
by
either
the
endogenous production of TNF-a (Green et al., 1990a; Sibley
et al.,
1991; Langermans et al., 1992) or by an exogenous
factor (such as LPS) that induces TNF-a production (Green et
al.,
1992).
MAC activation in the murine system has been
exaustively studied.
However, extrapolation to other species
has not always been successful (Douvas et al., 1986; Rook et
al., 1986; Toba et al., 1989; Krahenbuhl and Adams,
1994).
Activation of jird MACs was not accomplished using standard
in vitro methods routinely employed in the murine and other
systems.
Further, it was not possible to detect jird MAF in.
cultures
of
obtained
spleen
from
respectively.
cells
normal
stimulated
or
with
BCG-PPD
Con
A
or
immunized
PPD
jirds,
Parallel in vitro experiments in mice resulted
in MAC activation indicating that these methods and reagents
are effective in our laboratory.
immunization
activated
jird
However,
and
murine
in vivo BCG-PPD
MACs
to
kill
Toxoplasma cells.
The reasons why jird MACs could not be activated in
vitro
to
kill
Toxoplasma
via
MAF
are
at
this
moment
uncertain, but may be related to the inability of the jird to
express significant amounts of IFN-y.
Jird IFN-y mRNA was
not detected in lymph nodes and spleens from uninfected or B.
118
pahangi-infected jirds and IFN-y gene expression in Con A
stimulated
spleen
cells
was
extremely
low
(Mai,
1996).
However, this does not explain why jird MACs can be activated
in vivo but not in vitro.
It is possible that jird IFN-y or
other equivalent jird MAF becomes inactivated easily under
certain
conditions
in
vitro
or
that
unknown
inhibiting
substances in culture conditions have specific effects on
jird MAF.
Human MACs treated with combinations of IFN-y plus
TNF-a or LPS are unable to decrease survival of Mycobacterium
leprae
(Krahenbuhl and Adams,
1994)
or other mycobacteria
(Douvas et al., 1986; Rook et al., 1986; Toba et al., 1989)
as compared to mouse MACs (Krahenbuhl and Adams, 1994; Alfes
et al., 1985; Rook et al., 1986).
of
human MACs
in
vitro can
However, similar treatment
accomplish
killing
of
other
different intracellular organisms (Murray and Cartelli, 1983;
Hoover et al., 1985; Douvas et al., 1986).
These differences
in cell response between and within animal species indicate
that the signaling networks that lead to MAC activation may
be more complex than was first believed.
Furthermore,
the
fact that the NO pathway cannot be demonstrated consistently
in human MACs (review in Denis, 1994) has also raised doubts
about the concept of MAC activation defined in the murine
system.
The
NO
pathway has
been
implicated
as
primary
effector mechanism mediating cytotoxicity of activated MACs
(Nathan and Hibbs,
1991).
NO production was not found in
cultures of LPS-stimulated and non-stimulated,
peritoneal
119
MACs from control or BCG-PPD immunized jirds or in jird MAC
monolayers treated with a combination of Con A MAF and LPS.
However, parallel experiments with murine MACs demonstrated
NO generation as previously reported by other investigators
(Hibbs et al., 1987; James and Glaven, 1989; Adams et al.,
1990; Green et al., 1990b).
It is possible that the differences seen between mouse
and jird MACs to become toxoplasmacidal are due to variations
in susceptibility to this protozoan.
Results from in vivo
BCG-PPD immunization suggest that mouse MACs have a higher
capacity to restrict Toxoplasma growth than jird MACs.
Other
authors have found that although both mice and jirds appear
to be equally susceptible to the highly virulent RH strain of
Toxoplasma, the jird dies or develops severe clinical illness
when
infected
with
moderate
or
low
virulence
strains.
Whereas in the mouse these strains induce a chronic latent
infection (Suzuki and Tsunematsu, 1974) that results in the
presence of highly activated MACs
1968).
Interestingly,
(Ruskin and Remington,
humans and rats have a remarkable
resistance to Toxoplasma infection (Lainson, 1955).
Normal
human and rat alveolar MACs can kill Toxoplasma in vitro and
this microbial activity is oxygen-independent (Catterall et
al., 1986) suggesting that the particular resistance of these
species is due to the presence of a powerful non-oxidative
antimicrobial mechanism that could innately be turned on.
The requirements to trigger microbicidal mechanisms may vary
120
widely with animal species and specific pathogens which would
explain
the
differences
we
found
in
the
mouse
and
jird
systems of MAC activation.
Peritoneal MACs from jirds infected with female or male
B. pahangi were toxoplasmacidal at 15 DPI, but not at 50 DPI
and 135 DPI.
MAC activation in early B. pahangi infection
coincided with the peak in granulomatous reactivity around
parasite antigen-coated beads reported in chapter 2.
Lack of
toxoplasmacidal activity corresponded to downregulation of
the pulmonary granulomatous response.
No difference in MAC
microbicidal activity was demonstrated between female or male
B. pahangi infections, indicating that the presence of MF was
not required for MAC deactivation.
Similarly,
data from
chapter 2 indicate that downregulation of the PGRN is not
dependent
on
the
presence
of
MF.
The
increase
of
Th2
cytokine messages, mRNA IL-4 and IL-10, in cells from spleen
and lymph nodes of subcutaneously B. pahangi-infected jirds
(Mai,
1996)
occurs
at
the
same
deactivation in our experiments.
time
periods
than
MAC
A state of filarial immune
hyporesponsiveness with absence of marked inflammation has
also been associated with a shift to the Th2 cell phenotype
in humans
(King et al.,
1993; Maizels et al.,
1995).
The
lack of toxoplasmacidal activity of MACs in chronically B.
pahangi-infected jirds may be related to the presence of Th2
cytokines that deactivate MACs, such as IL-4
1989; Ho et al.,
1992) and IL-10
(Lehn et al.,
(De Waal Malefyt et al.,
121
1991;
De Waal-Malefyt et al.,
1991;
Silva et al.,
1991).
These cytokines exert a negative effect on proinflammatory
molecules which would explain the depressed PGRN found in our
experiments.
Female worm infection resulted in high accumulation of
peritoneal MACs presumably due to the continuous release of
MF that acted as a potent inflammatory stimulus (chapter 2).
Interestingly,
these
MACs
were
not
Toxoplasma and rarely formed granulomas.
activated
to
kill
As it has shown in
diverse intracellular organisms (review in Reiner, 1994) , the
interaction MAC-parasite causes defects on the MAC effector
functions that may result in suppressive effects on other
immune cells.
IL-10,
For instance, MACs can be induced to secrete
TGF-/3 and prostaglandin E2 (PGE2) that downregulate
cell-mediated immunity and may drive the immune response to
a Th2 phenotype.
Molecules from filarial nematodes that may
exert a direct effect of MAC function have not been well
studied.
However, MF have been shown to release PGE2 (Liu et
al., 1992) . This inflammatory molecule has been demonstrated
to be a potent immune modulator suppressing MAC (Taffet and
Russell, 1980; Snyder et al., 1982) and lymphocyte functions
(Ellner and Spagnuold, 1986).
Previous studies demonstrated that MACs from B. pahangiinfected jirds with chronic infections were activated to kill
S. aureus (Jeffers et al., 1984).
These results may differ
from the current data because the stage to initiate infection
122
were L3, which in the chronic phase resulted in greatest MAC
accumulation with characteristic granuloma formation (Jeffers
et
al.,
1987).
On
the
other
hand,
the
immunological
requirements to kill Toxoplasma cells may vary from those to
kill S.aureus.
Microbicidal activity to kill S.aureus of
MACs from L3 B.pahangi-infected jirds was similar to that of
thioglicolate-elicited control MACs.
However,
attempts to
obtain toxoplamacidal activity in thioglicolate-elicited MACs
failed
(data not shown).
probably
requires
Killing of Toxoplasma organisms
different
immune-mediated
signals
killing of facultative organisms such as S. aureus.
than
Other
investigators, however, have found variations in the ability
of MACs to cope with organisms of more similar background.
For
instance,
it
has
been
found
that
susceptibility
of
Leishmania to be killed by presumably activated MACs varies
markedly upon species and stages of the parasite (Murray and
Cartelli, 1983; Hoover et al., 1985).
Increase in spontaneous and LPS-induced TNF production
above that of controls occurred at 15 DPI in both female and
male worm infections corresponding to MAC activation.
LPS-
induced TNF production peaked at 56 DPI and decrease markedly
at 135 DPI in both female and male infections.
The peak in
TNF production occurred at the moment MACs were accumulating
in large numbers, especially in the female infection; and may
be
related
cytokine.
to
the
potent
chemotactic
function
of
this
The subsequent decrease could have been induced by
123
similar factors that caused MAC deactivation.
It has been
demonstrated that deactivating cytokines such as IL-10 and
IL-4 inhibit production of TNF-a (Hart et al., 1989; De Waal
Malefyt et al., 1991). These cytokines may be more abundant
or may exert a more intense downregulatory effect as the
infection progresses to the chronic time period.
IL-4 and
IL-10 mRNA in B. pahangi-infected jirds are highest at 150
DPI (Mai, 1996).
Further work is needed in the jird model to discern the
factors and mechanisms involved in the downmodulation of the
inflammatory
and
immune
responses
to
filariae.
We
demonstrated that the parasite-specific hyporesponsive state
defined in the jird infected with B. pahangi is associated
with
a defect
in MAC
function that
is manifested
incapacity to kill Toxoplasma and to produce TNF-a.
as
an
The MAC
is recruited locally in large numbers in response to filariae
infection and may prove to be a key effector cell implicated
in the immunoregulatory mechanisms that determine the disease
outcomes in human filariasis.
CHAPTER 5.
CONCLUSIONS
Lymphatic filariasis is a human parasitic disease with
a spectrum of clinical conditions that reflect the intensity
and pattern of immune responses to the parasite
Nutman,
1991).
The
two principal poles of
(King and
the clinical
states are asymptomatic microfilaremia and amicrofilaremic
chronic pathology.
The jird-B. pahangi model has been extensively used to
study immunopathogenesis of filariae and has been proved to
be
a suitable
experimental
system to investigate
factors
implicated in filarial immunomodulation and pathology.
The
jird intraperitoneally infected with different stages of B.
pahangi
was
used
to
further
characterize
the
stage
specificity of the inflammatory response and the role of the
MAC in the downmodulation of this response.
The second chapter addressed the effect of different B.
pahangi
developmental
different
systemic
parasite
stages
numbers
inflammatory
as well
on
the
response.
as dead worms,
downmodulation
Results
of
indicated
and
the
that
viable worms are required to induce downregulation of the
inflammatory response, and this downregulation occured in the
absence of MF.
Inoculation of male, female, L4, L3, and MF
resulted in decreased PGRN as the infection progressed to the
chronic
period.
Higher
female
worm
burdens
induced
a
significantly greater and more rapid PGRN decrease than low
124
125
burdens.
PGRN decrease induced by adult worms was greater
than that induced by MF.
Therefore, data also indicated that
parasite burden and probably chronicity effected the state of
filarial hyporesponsiveness.
Experiments described in chapter 3 were conducted to
better
discern
the
downmodulation of
role
the
of
PGRN.
developing
Jirds
larvae
were
in
the
inoculated with
normal L3 or with L3 irradiated with increasing levels of
gamma radiation.
Gamma radiation inhibited the growth and
decreased the survival of B. pahangi as previously reported
(Oothuman et al., 1978; Devaney et al., 1993).
Decrease in
parasite survival and worm lengths was inversely related to
the levels of irradiation as observed by Oothuman et al.
(1978).
doses
Male
Some parasites irradiated with the lowest radiation
(15 and 25 krads) developed into adult female worms.
worms
were
Downmodulation
not
of
the
found and
females
PGRN did not
were
occur
not
in
gravid.
jirds
received L3 irradiated with more than 15 krads.
that
However,
normal L3 and L3 irradiated with 15 krads induced significant
PGRN decrease at the chronic time period.
support
the
importance
downregulation of
prove
again
that
downregulation.
the
MF
of
the
systemic
are
adult
These results
stage
in
inflammatory response
not
needed
to
induce
the
and
this
It is not clear whether L4 are important in
the downmodulation of the PGRN.
However, the fact that PGRN
decrease occured at 28 DPI when the molt to adults had just
126
occurred,
suggested
downregulation.
that
L4
do
induce
some
level
of
The absence of PGRN downregulation in jirds
infected with L3 exposed to radiation doses higher than 15
krads may be explained by the low total parasite burden, the
absence or minimal numbers of adult worms, or by the effect
of
high
radiation
on
the
release
of
specific
parasite
products.
Experiments described in chapter 2 and 3 showed that the
peritoneal inflammatory response, measured by MAC numbers,
was greatest
burdens.
in jirds with persistent
intraperitoneal MF
This indicated that the release of MF constitute a
potent
inflammatory
stimulus
in
situations
compartmentalization may occur, and that this
stage may
contribute
elephantiasis cases.
to
the
where
developmental
lymphatic pathology seen
in
Formation of peritoneal granulomas in
jirds with normal L3 infections occurred more frequently at
the chronic time period when MF were produced.
Peritoneal
granulomas were found less often in infections with female
worms.
in
This may be explained because MF numbers were lower
these
latter
infections.
Alternatively,
the
early
presence of adult worms may prevent formation of peritoneal
granulomas by downmodulatory mechanisms similar to those that
decreased the PGRN,
but still allowed the recruitment of
considerably number of MACs due to the presence of parasite
products and/or MF.
Infection with irradiated parasites
resulted in frequent formation of peritoneal granulomas at 7
127
and 14 DPI, when compared to the paucity of granulomas at
these time points following infections with normal L 3 . Dead
and damaged larvae were probably more abundant shortly after
infection with irradiated L3 and presumably were more capable
of inducing an inflammatory reaction.
Irradiated parasites
that survived probably were not damaged and less effective at
inducing local inflammation.
Chapter 4 described the studies on MAC function in jirds
infected IP with female or male B. pahangi.
The microbicidal
activity against Toxoplasma, as well as NO and TNF production
were used as indicators of MAC activation.
Prior
to
experimental
filarial
infection,
jird
MAC
activation was characterized by comparing it to mouse MACs.
Attempts to confer toxoplasmacidal activity to MACs in vitro
with a combination of murine rlFN-y, Con A MAF or BCG-PPD MAF
and LPS succeeded in the mouse but failed in the jird.
In
vivo immunization with BCG-PPD yielded positive results in
both species.
However, the toxoplasmacidal activity of mouse
MACs appeared to be greater than that of jird MACs after BCGPPD
immunization,
which
may
be
related
to
a
higher
susceptibility of jirds to Toxoplasma as other studies have
shown (Suzuki and Tsunematsu, 1974).
NO production by jird
MACs could not be demonstrated after in vitro or in vivo
treatments to activate MACs.
MAC activation occurred in jirds infected with female or
male B. pahangi at 15 DPI, but not at 50 and 135 DPI.
TNF-
128
like production increased at 15 DPI in both, female and male
worm infections corresponding to MAC activation.
LPS-induced
TNF production peaked at 56 DPI and decrease markedly at 135
DPI
in
both
female
and
male
worm
infections.
MAC
deactivation and decrease of TNF levels coincided with the
downregularion of the PGRN.
These data indicate that the
factors responsible for the absence of MAC activation and
decrease
of
TNF
levels
observed
during
chronic
Brugia
infection may be related to the mechanisms that induce the
downmodulation of the systemic inflammatory response.
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VITA
Carmen Nasarre,
the daughter of Antonio Nasarre
and
Montserrat Azara, was born in Barcelona, Spain, on October
26,
1967.
She obtained her Doctor of Veterinary Medicine
degree from the Faculty of Veterinary Medicine, University of
Zaragoza, Spain, in September,
1990.
Her determination to
pursue a career in Pathology brought her to Louisiana in
August,
1991 by mediation of Dr.
Enright and Dr. Casey.
department
Jose Maria Blasco,
She started her PhD program in the
of Veterinary Pathology where
pathology residency.
to work in lymphatic
Dr.
she completed a
In June, 1993 she joined Dr. Klei'lab
filariasis.
She
then became a PhD
candidate in the department of Veterinary Microbiology and
Parasitology.
153
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
M aj or Field:
Carmen Nasarre
Veterinary Medical Sciences
Inflammatory Responses of the Jird
to Brugia pahangi: Parasite Stage Specificity and Role of
the Macrophage
Title of Dissertation:
Approved:
Major Professor and Chairman
f
Deah
aha of tnB Graduate School
EXAMINING^COMMrETEE
Date of Examination:
February 29, 1996