The importance of mucosal mast cells in the host response to Hymenolepis diminuta in normal, W/Wv,
and nude mice
by Patti Kay Havstad
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biological Sciences
Montana State University
© Copyright by Patti Kay Havstad (1987)
Abstract:
The response of normal mice to the tapeworm, Hymenolepis diminuta, is characterized by an
accumulation of intestinal mucosal mast cells (MMC) and eosinophils and a limited growth and
lifespan of the adult worm. In comparison, athymic nude mice respond to the tapeworm with intestinal
eosinophil, but no MMC accumulation, and the worm's growth and lifespan are extended. To evaluate
the involvement of the MMC and the influence of the thymus in the host response to H. diminuta, three
cysticercoids of the parasite were given to normal littermate (LM), mast cell deficient W/Wv > mast
cell repaired W/Wv (W/WvBM), and semi-syngeneic C57BL nude mice. LM mice rejected the parasite
by day 10 PI while it was retained by W/Wv and W/WvBM mice up to day 21 PI and by C57BL nude
mice up to day 16 PI. The length and weight of worms recovered were substantially greater in W/Wv
and W/WvBM mice than they were in either LM or nude mice. MMC accumulated in response to Hi
diminuta in the intestines of LM and WZWvBM mice but not in the intestines of unrepaired WZWv
mice, and occurred in unexpectedly high numbers in the intestines of both C57BL and BALB/c nudes
while eosinophils increased significantly in the intestines of all mice except C57BL and BALBZc
nudes. The peripheral blood leukocyte response was highly variable (in all mice), yet peripheral blood
eosinophils appeared to have a pattern of increase which occurred prior to observed increases of
intestinal eosinophils. The intermediate host source of H. diminuta, either Ti molitae or Ti confusum,
may have affected the time courses of worm growth and intestinal MMC and eosinophil increases, but
did not appear to affect the overall growth of the worm or the time of its rejection. It was concluded
that MMC play a functional, but not essential role in the host response to Hi diminuta and that athymic
nude mice respond less efficiently to Hi diminuta than do normal littermates, but more efficiently than
do either mast cell-deficient or mast cell-repaired W/Wv mice. THE IMPORTANCE OF MUCOSAL MAST CELLS IN THE HOST RESPONSE
TO HYMENOLEPIS DIMINUTA IN NORMAL, W/WV , AND NUDE MICE
by
Patti Kay Havstad
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Biological Sciences
MONTANA STATE UNIVERSITY
Bozeman, Montana
June 1987
MAIN LIB.
ii
APPROVAL
of a thesis submitted by
Patti Kay Havstad
This thesis has been read by each member of the thesis committee
and has been found to be satisfactory regarding content, English usage,
format, citations, bibliographic style, and consistency, and is ready
for submission to the College of Graduate Studies.
Date
g/rA?_____
'
'
Zr/Lwf,
Chairperson, Graduate Committee
Approved for the Major Department
Date
Head, Major Department
Approved for the College of Graduate Studies
2
Date
j-f 7
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In
presenting
this
thesis
in
partial
fulfillment
of
the
requirements for a master's degree at Montana State University, I agree
that the Library shall make it available to borrowers under rules of
the Library.
Brief quotations from this thesis are allowable without
special permission, provided that accurate acknowledgment of source is
made.
Permission for extensive quotation from or reproduction of this
thesis may be granted by my major professor, or in her absence, by the
Dean of Libraries when, in the opinion of either, the proposed use of
the material is for scholarly purposes.
Any copying or use of the
material in this thesis for financial gain shall not be allowed without
my written permission.
Signature
Date
iv
ACKNOWLEDGMENTS
I thank my major advisor,
Dr.
Patricia
guidance and assistance with this work,
graduate committee,
Speer,
Dr.
Burgess,
Dr.
K.
her
and the other members of my
Paden, Dr.
for their valuable time and suggestions.
acknowlege
Crowle, for
the contributions of Norman D.
Phillips,
and
Dr.
I also gratefully
Reed of the Microbiology
Department for his advice on the subject matter of this thesis, Martin
Hamilton of the Mathematics Department
statisitics, and
Gayle
for his assistance with
Callis of the Veterinary Science Histology
Laboratory for her assistance with histotechnique.
to my husband,
Kris,
the
and children,
constant support of this endeavor.
Special thanks go
Jonathan and Jessica,
for their
V
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ................ ..........................
iv
LIST OF TABLES .......................................... .
vii
LIST OF FIGURES ............ ..............................
xi
ABSTRACT ........... ............................ .........
xi
INTRODUCTION............................. .... ..........
I
LITERATURE REVIEW ................ •............. .........
5
Functional activity of stimulated mast cells ....... .
Heterogeneity, fixation, and staining ..............
Origin, differentiation, and development ........... .
Interaction with IgE ..........................
Interaction with eosinophils ........ ...............
Parasite and host influence ........................
Animal models ................................... . •
MATERIALS AND METHODS .... .................... ..........
Mice ..................... .........................
Bone marrow repair ..... ............................
Parasite .......... ..................... ...........
Experimental protocol ..............................
Littermate, WZWv f WZWv bone marrow repaired mice
Intermediate host: Tenebrio m o l i t a e .... .......
Littermate and WZWv mice
Intermediate host: Tribolium confusum.........
Nude mice ........... ...............*..........
Worm recovery ................................. .....
Worm measurement ........................ ...........
Histology .................................. ........
.Blood cell counts .................. .......... .....
PCA assay for worm-specific IgE ...... ..............
Statistical analysis' ..... ..........................
9
11 •
13
19
20
21
22
27
27
27
28
29
29
30
30
31
32
33
35
36
37
RESULTS ....................... ...............................
39
Littermate, WZWv , and WZWv bone marrow repaired mice
Intermediate host: Tenebrio molitae .............. .......
Worm recovery ............... *......................
Worm growth ...... ..................................
39
39
vi
TABLE OF CONTENTS (continued)
Histological observations ......
Blood cell response ........ .......... ..........
Worm specific IgE ...................................
Littermate and W/WV mice
Intermediate host: Tribolium confusum . ........ ...........
Worm recovery ............
Worm g r o w t h .... ................
Histological observations ...............
Tenebrio molitae vs. Tribolium confusum. Intermediate
hosts .......................'........................
Nude mice .....................
Worm recovery .......................................
Worm growth ......................
Histological observations ......... *................
Blood cell response .....
Worm specific IgE ...................
45
49
51
57
61
61
63
66
72
72
DISCUSSION ................................... .................
. 76
54
54
55
57
SUMMARY ...............
88
REFERENCES ...............................
92
APPENDIX
105
vii
LIST OF TABLES
Table
1.
2.
3.
4.
5.
6.
Page
Percentage of mice positive for adult worms
POS) and
percentage of cysticercoids recovered as adult worms (% REC)
at different intervals post-inoculation (PI) of littermate,
W/Wv , W/Wv bone marrow repaired (WZWvBM), and control C57BL
nude (C57BL NU) mice with 3 cysticercoids of
diminuta ....
40
MMCZ10 VCU in four sections of the small intestine of
representative
diminuta infected littermate mice show a
small degree of regional differences ..................... ..
45
Differential leukocyte counts of 100 cells in Wright's stained
blood.smears made from blood collected from LM, WZWv , and
WZWvBM mice on alternate days post-inoculation and starting on
day -I ..... .................................... ........ .
53
Passive cutaneous anaphylaxis tests for the presence of wormspecific IgE in the sera of littermate (LM)1 WZWv , and WZWv
bone marrow repaired (WZWvBM) mice collected on days 16, 21,
and 28 post-inoculation (PI) with 3 cysticercoids of H.
diminuta ......................................... .........
54
The percentage of mice positive for adult worms (% PCS) and
the percentage of injected cysticercoids recovered as adult
worms {% EEC) in littermate (LM) and WZWv mice with the
• intermediate host source Tribolium confusum.................
Probabilities (p values) of the significant difference between
the response of littermate (LM) and WZWv mice to H. diminuta
derived from two different intermediate host sources; Tenebrio
molitae and Tribolium confusum.......
55
60
7.
Percentage of mice positive for adult worms {% POS) and
and percentage of cysticercoids recovered as adult worms {% REG)
at different intervals post-inoculation (PI) of C57BL littermate
(LM), CS7BL nude (NU), BALBZc littermate (LM), and BALBZc nude
(NU) mice...................................................
62
8.
PCA tests for the presence of worm-specific IgE in the serum of
C57BL littermate (LM), C57BL nude (NU), BALBZc littermate (LM)
and BALBZc nude (NU) mice on day 21 post-inoculation (PI) with
H. diminuta...............
75
viii
LIST OF TABLES (continued)
9.
Mean values for worm length, weight, mucosal mast cells (MMC)
per 10 villus crypt units (VCU) and eosinophils (EOS) per 10
VCU in littermate (LM), W/Wv , W/Wv bone marrow repaired
(WZWvBM) and control CS7BL nude mice on days 0, 6/7, 10, 12,
16, 21, and 24 post inoculation with 3 cysticercoids of H.
diminuta......... ..........................................
106
10. Eosinophils per cubic millimeter of peripheral blood collected
from littermate (LM), WZWv , WZWv bone marrow repaired (WZWvBM),
and control C57BL nude (CS7BL NU) mice on alternate or
specific days post-inoculation (PI) with 3 cysticercoids of
diminuta......... ...... ...................................
10&
11. Mean values for worm length, weight, mucosal mast cells (MMC)
per 10 villus crypt units (VCU), and eosinophils (EOS) per 10
VCU in littermate and WZWv mice on days 6, 12, 18, and 24 postinoculation (Pl) with 3 cysticercoids of H1. diminuta,
intermediate host: Tribolium confusum.......... .............
109
12. Mean values for worm length, weight, mucosal mast cells (MMC)
per 10 villus crypt units (VCU), and eosinopils (EOS) per 10
VCU in C57BL littermate (LM) and nude (NU) mice on days 0, 7,
12, 16, and 21 post inoculation (PI) with 3 cysticercoids of
H. diminuta ............................... ................
110
13. Mean values for worm length, weight, mucosal mast cells (MMC)
per 10 villus crypt units (VCU), and eosinophils (EOS) per 10
VCU in BALBZc littermate (LM) and nude (NU) mice on days 0, 7,
12, 16, and 21 post-inoculation (PI) with 3 cysticercoids of .
H. diminuta....... ......................................... •
111
14. Eosinophils per cubic millimeter of peripheral blood collected
from C57BL littermate (C57BL LM), C57BL nude (C57BL NU),
BALBZc littermate (BALBZc LM), and BALBZc nude (BALBZc NU)
mice on alternate days post^inoculation (PI) with 3
cysticercoids of IL_ diminuta .............. "......
112
ix
LIST OF FIGURES
Figure
Page
I.
Mean length (cm) of
diminuta worms recovered from littermate,
W/Wv , WZWv bone marrow repaired, and control CS7BL nude mice
at different intervals post-inoculation ............ ........
42
2.
Mean weight (mg) of Hi. diminuta worms recovered from littermate,
W/Wv , WZWv bone marrow repaired, and control CS7BL nude mice
at different intervals post inoculation .............. ......
43
3. . Photograph of Hi. diminuta worms recovered from littermate (LM),
WZWv , and control CS7BL nude (BLN) mice on days 6, 10, 16, and
21 PI.............. .........................................
4.
5.
6.
7.
Mean number
units (VCU)
bone marrow
inoculation
44
of mucosal mast cells (MMC) per 10 villus crypt
in the intestines of littermate, WZWv , and WZWv
repaired mice at different intervals postwith 3 cysticercoids of Hi. diminuta .............
47
Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of littermate, WZWv , WZWv bone marrow
repaired* and control CS7BL nude mice at different intervals
post-inoculation with 3 cysticercoids of Hi. diminuta ........
48
Mean number of eosinophils (EOS) per cubic millimeter (mm3) of
peripheral blood collected from littermate, WZWv , and WZWv bone
marrow repaired mice on alternate days post-inoculation with
3 cysticercoids of Hi. diminuta............................... .
50
Eosinophils (EOS) per cubic millimeter (mm3) of peripheral
blood collected from a single representative littermate. mouse
on alternate days post-inoculation with 3 cysticercoids of Hi.
diminuta........ ...................... ....................
52
8.
Mean length (cm) and weight (mg) of Hi. diminuta worms recovered
littermate and WZWv mice at different intervals post­
inoculation with 3 cysticercoids derived from the intermediate
host T.confusum -........ ....................................
56
9.
Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of littermate and WZWv mice at
different intervals post-inoculation with 3 cysticercoids of
H. diminuta derived from the intermediate host T i. confusum ....
58
X
LIST OF FIGURES (continued)
10. Mean number of eosinophils per 10 villus crypt units (VCU) in
the intestines of littermate and W/Wv mice at different
intervals post-inoculation with 3 cysticercoids of H l diminuta
derived from the intermediate host T. confusum ...............
59
11. Mean length (cm) of H l diminuta worms recovered from C57BL
littermate, C57BL nude, BALB/c littermate, and BALB/c nude mice
at different intervals post-inoculation ................ ....
64
12. Mean weight (mg) of H l diminuta worms recovered from CS7BL
littermate, C57BL nude, BALB/c littermate, and BALB/c nude mice
at different intervals post-inoculation .....................
65
13. Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of CS7BL littermate and nude mice
at different intervals post-inoculation with 3 cysticercoids of
H. diminuta .................... .............................
67
14. Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of BALB/c littermate and nude
mice at different intervals post-inoculation with 3
cysticercoids of Hl diminuta.................................
68
15. Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of CS7BL littermate and nude mice at
different intervals post-inoculation with 3 cysticercoids of
H. diminuta .................................................
70
16. Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of BALB/c littermate and nude mice at
different intervals post-inoculation with 3 cysticercoids of
Hl diminuta ................. ...............................
71
17. Mean number of eosinophils (EOS) per cubic millimeter (mm3) of
peripheral blood collected from C57BL littermate and nude mice
on alternate days post-inoculation with 3 cysticercoids of Hl
diminuta ................................ ............. ......
73
18. Mean number of eosinophils (EOS) per cubic millimeter (mm3) of
peripheral blood collected from BALB/c littermate and nude mice
on alternate days post-inoculation with 3 cysticercoids of Hl
diminuta .....................................................
74
xi
ABSTRACT
The response, of normal mice to the tapeworm, Hymenolepis
diminuta, is characterized by an accumulation of intestinal mucosal
mast cells (MMC) and eosinophils and a limited growth and lifespan of
the adult worm. In comparison, athymic nude mice respond to the
tapeworm with intestinal eosinophil, but no MMC accumulation, and the
worm's growth and lifespan are extended. To evaluate the involvement of
the MMC and the influence of the thymus in the host response to H l
diminuta, three cysticercoids of the parasite were given to normal
littermate (LM), mast cell deficient W/Wv > mast cell repaired W/Wv
(WZWvBM), and semi-syngeneic C57BL nude mice. LM mice rejected the
parasite by day 10 PI while it was retained by WZWv and WZWvBM mice up
to day 21 PI and by C57BL nude mice up to day 16 PI. The length and
weight of worms recovered were substantially greater in WZWv and WZWvBM
mice than they were in either LM or nude mice. MMC accumulated in
response to £L diminuta in the intestines of LM and WZWvBM mice but not
in the intestines of unrepaired WZWv mice, and occurred in unexpectedly
high numbers in the intestines of both C57BL and BALBZc nudes while
eosinophils increased significantly in the intestines of all mice
except C57BL and BALBZc nudes. The peripheral blood leukocyte response
was highly variable (in all mice), yet peripheral blood eosinophils
appeared to have a pattern of increase which occurred prior to observed
increases of intestinal eosinophils. The intermediate host source of H l
diminuta, either T l molitae or Tl confusum, may have affected the time
courses of worm growth and intestinal MMC and eosinophil increases, but
did not appear to affect the overall growth of the worm or the time of
its rejection. It was concluded that MMC play a functional, but not
essential role in the host response to H l diminuta and that athymic
nude mice respond less efficiently to H l diminuta than do normal
littermates, but more efficiently than do either mast cell-deficient or
mast cell-repaired WZWv mice.
I
INTRODUCTION
The mucosal mast cell (MMC) accumulates in the small intestine of
rodents in response to invasion by multicellular antigens including
several nematode and cestode parasites
(Taliaferro and Sarles,
Wells,
Gray,
1962;
Kelly and Oglivie, 1972;
Elgersma, 1976; Andreassen et al.,
1976;
1939;
Ruitenberg
and
1978; MacDonald et al., 1980; Lee
and Wakelin, 1982; Handlinger and Rothwell, 1984).
The role of the MMC
in the host response is not known, although several suggestions have
been made, such as involvement in the parasite rejection mechanism
(Kelly and Dineen, 1976; Harari et al., 1987).
Studies on the nature
of this response, even those with the same host and parasite species,
have yielded a variety of conflicting results.
It is clear that the
extent of the MMC response is dependent upon the integrity
of
the
hosts' immune system and the nature of the parasite stimulus.
Genetically athymic nude mice have often been used as a model to
evaluate the role of the thymus and MMC in parasite rejection (Isaak et
al.,
1975;
Ruitenberg and Elgersma, 1976;
Reed et al.,
rejection of parasites from nude mice is often delayed,
1982).
The
for example,
nude mice retain the cestode, H^ diminuta, up to three, months, compared
to an average retention of 10 days in normal mice (Issak et al., 1975).
The delayed rejection of parasites from nude mice may be due to the
lack of several thymus dependent factors, including the MMC (Ruitenberg
and Elgersma, 1976).
The
involvement
specifically
of
studied with
MMC
in
parasite rejection has been more
the W/Wv mouse model.
W/Wv mice have
a
2
genetic stem cell-related defect which affects their ability to produce
both connective tissue (CTMC) and M G
al.,
(Kitamura et al., 1978; Uber et
1980), while the immune response potential in terms of B and T
lymphocyte functions appears normal in these mice (Mekori, 1969; Ha et
al.*
1986).
Parasite rejection from
WZWv mice has been reported as
being considerably impaired (Crowle and Reed, 1981), slightly impaired
(Mitchell
et
infections
al.,
with
1983)
the
or
not
nematodes
Trichinella spiralis.
impaired
(Uber
et al.,
Nippostrongylus
1980)
brasiliensis
in
and
Correction of the stem cell-related defect with
bone marrow from normal littermates has been reported to have no effect
on
expulsion
of
brasiliensis
(Crowl.e,
1983)
but
to accelerate
expulsion of T l spiralis (Ha et al., I983)•
Use of the WZWv mouse system with Hl diminuta infections allows
evaluation of the MMC response to a different parasite stimulus.
Hl
diminuta is a noninvasive cestode which resides in the intestinal lumen
of
a
rodent
brasiliensis
definitive
and T l
host
(Befus, 1975) , in
spiralis which have
contrast
invasive larval
to
stages
Nl
and
exist as adults in intimate contact with the host enterocytes (Wright,
1976; Kassai, 1982).
H. diminuta infection of the definitive host is
- by ingestion (or inoculation) of the larval cysticercoid, which resides r
within a flour beetle intermediate host.
The ingested
cysticercoid
migrates to the proximal small intestine,
attaches to the intestinal
wall
(progressively
via
segments)
its
scolex,
(Owen, 1972).
and
strobilates
developes
The mechanism of rejection of Hl diminuta is
not known, but involves destrobilation of the worm (Hopkins et al. ,
1972)
and
may
be
dependent
upon the degree of direct
interaction
3
between the parasite (via the scolex) and the host tissue (Gray, 1976;
Andreassen et al., 1978).
The potential activity of mediators released from stimulated MMC,
such as histamine and prostaglandins, may contribute to the rejection
mechanism.
Histamine
and prostaglandins create alterations in the
intestinal environment,
secretion,
including
peristalsis
increased
fluid
which may be deleterious to parasite establishment
(Kelly
and Dineen, 1976; Harari et al., 1987).
and
Other mast cell mediators are
chemotactic for components of the host protective system, such as the
eosinophil, which may provide additional effector functions in parasite
rejection (Kay, 1979) •
Eosinophilia
infections
1979;
is
a well documented characteristic of parasite
(Oglivie, 1964;
K a y , 1 979;
Dessein
eosinophils and MMC has
Ruitenberg et al., 1977;
et
been
al.,
1981).
reported
Hasten et
al. ,
A relationship between
at many
levels,
including
histological (Wells, 1965; Mann, 1965) and cytochemical (Capron et al.,
1978).
The
activation,
two cell
since
types are not
dependent upon each
other
for
eosinophilia has been reported to occur in both
normal and athymic nude mice in response to H l diminuta (Andreassen et
al., 1978; Hindsbo et al., 1982), but the mutual presence of MMC and
eosinophils may enhance the protective response of the host (Capron et
al., 1978; Kay, 1979; Henderson et al., 1980).
Elevation in the level of serum IgE is also a well documented
characteristic of parasite infections (Oglivie, 1964; Ishizaka et al. ,
1976; Mayrohofer et al., 1976; Jarret and Path 1973; Hindsbo et al.,
1982), however, in the case of H l diminuta infected mice, the level of
4
parasite-specific IgE in the sera is apparently not
1977).
Since mast
elevated
cells serve as a main repository
(Befus,
for IgE in the
tissues (Mayrohofer et al., 1976) a minimal amount of specific IgE may
be produced which remains bound to the cell surface and, therefore, is
not detectable in the sera (Nawa and Miller,
mediators from mast
1979) •
The release of
cells through a mechanism of (surface-bound) IgE
cross-binding with specific antigen
1972) suggests
(Ishizaka et al.,
that IgE production is an integral part of the mast cell response and
likely occurs even when not detectable by a standard IgE assay.
In this study the influence of MIC on the growth and rejection of
the rodent tapeworm, iL_ diminuta, was evaluated using W/WV mast celldeficient mice
peripheral
and their mast
blood eosinophil
were also evaluated.
tests.
Athymic
comparative purposes,
littermates.
Tissue
and
as well as the total leukocyte responses
The presence of Hj_ diminuta-specific IgE in the
sera of infected mice was
(PCA)
cell-normal
assayed
nude
mice
by passive
were
cutaneous
included
anaphylaxi s
in this
study
for
to assess the thymus and MMC influence on the
host response, and to provide parasite-positive controls. Additionally,
the influence of the intermediate host, flour beetles
Ti molitae or T.
confusum, on the dynamics of the infection was compared.
5
LITERATURE REVIEW
Rodent mast cells exist as two populations, the mucosal mast cell
(MMC)
and the connective tissue mast
cell
(CTMC).
These two cell
subtypes share many characteristics, such as localization in areas of
the
body
which
come
(Askenase, 1980;
into
contact
with
Crowle and Phillips,
1984) , characteristic
metachromatic
the
1983;
external
Wingren
and
environment
Enerback,
staining of their cytoplasmic
granules (Enerback, 1966b, Mayrohofer, 1980), surface affinity for IgE
(Izhizaka et al., 1972; Mayrohofer et al, 1979; Askenase, 1980), bone
marrow origin from a common stem cell (Kitamura, 1979b; Kitamura, 1981;
Crowle and Reed, 1984) and response to certain antigens by release of
granule mediators
(Murray et al., 1971; Wingren and Enerback,
1984).
These two cell subtypes differ in respect to the specific tissue in
which they locate
(Crowle and Phillips,
(Crowle and Phillips,
1983),
chemical
1983),
cellular morphology
composition of their granules
(Miller and Walshaw, 1972; Tas and Berndsen, 1977; Mayrhofer, 1980) ,
the nature of the stimuli to which they respond (Enerback and Lundin,
1974; Pearce et al., 1982), and dependence on a thymus influence for
development (Ruitenberg and Elgersma, 1976; Mayrohofer,. 1979; Reed et
al., 1982),
parasite
MMC accumulate in the
infections
Crowle and Reed,
small intestine in response to
(Miller and Jarret, 1971;
Murray
et al., 1971;
1981) and CTMC are distributed throughout the body
with concentrations in the skin and around blood vessels (Crowle and
Phillips, 1983).
The MMC is smaller than the CTMC and has fewer and
more irregular cytoplasmic granules (Crowle and Phillips, 1983)•
more sensitive to fixation and staining, requiring specific
It is
techniques
6
for
demonstration in the tissues (Enerback, 1966a, Enerback, 1966b;
Miller and Walshaw,
1971; Crowle and Phillips,
1983).
Additionally,
the MMC is under the influence of thymus educated !-lymphocytes for
growth
and
proliferation
heterogeneity
of the
two
(Ruitenberg
subtypes
and Elgersma, 1976).
of mast
cells
assumption that they perform different functions.
differentiation
into
two
distinct
subtypes
has
The
led to
the
The level at which
occurs
is
unknown
(Mayrhofer, 1979; Askenase 1980; Kitamura et al., 1981).
Mast cells may have evolved specifically to provide protection
against parasite infections (Kay, 1979; Kaliner, 1979; Saavedro-Delgado
et al., 1984).
The location of MMC and CTMC at sites
where parasites
frequently gain access to host tissue supports this hypothesis.
accumulate in response to many
intestinal
parasites,
MMC
including
a
variety of cestodes and nematodes (Gray, 1976; Andreassen et' al., 1978;
Ruitenberg and Elgersma, 1976; Crowle and Reed, 1981). ,
The MMC response is panmucosal and nonspecific (Jarret and Path,
1973; Wakelin, 1978; Pitts and Mayrohofer, 1983) which is reflected in
its involvement in allergic reactions.
Clincal allergy may result from
the activity of mast cells responding to an excessive insult or the
inablity of mast
cells to distinguish the antigenic determinants on
complex allergens
(allergy causing environmental antigens) from those
on
parasites
(Lewis
Wasserman, 1984).
and
1981;
Barret and Metcalf,
1984;
CTMC accumulate in response to cutaneous parasites,
including ectoparasites
larvae
Austin,
(Matsuda et al.,
(Talifarro and Sarles, 1939;
Hsu
1985) and skin penetrating
et
al.,
1979).
CTMC
are
associated with other functions, such as tumor angiogensis, by virtue
7
of their proximity to blood vessels (Schittek et a l ., 1985).
The
mucosal
mast
cell
response
was
first
reported
in 1939
(Taliaferro and Sarles) in an histological study of the intestines of
rats infected with
brasiliensis (N. muris).
MMC accumulation , up
to 7-8x normal (non-infected), (Murray et al., 1971) was subsequently
reported
in response
to other parasites in several mammalian hosts,
including humans (Gustowski et al., 1983)•
Most of the studies on the
MMC response have utilized intestinal nematodes, notably T^ spiralis
and
brasiliensis, and have been done in rat and mouse hosts.
The
peak
of
MMC
accumulation
has been
reported to occur in
various temporal associations with parasite rejection,
the parasite and the host.
depending upon
In N l brasiliensis infected rats, peaks of
MMC accumulation have been reported to occur at the same time as worm
rejection
(Miller and Jarret, 1971;
Murray et al.,
Miller, 1979: Woodbury et al., 1984) or after
1962; Keller,
1971; Nawa and
worm rejection (Wells,
1971; Kelly and Oglivie, 1972; Mitchell et al.,
1983).
Studies with Tl spiralis in rats indicate that MIC accumulation occurs
primarily at the time of rejection (Wells 1962, Woodbury et al., 1984;
Alzideh and Murrell, 1984).
In mice, MMC have been reported to occur
in peak numbers during (Crowle and Reed, 1981; Ruitenberg and Elgersma,
1976) and after (Uber et al.,
1980,
Alzideh and Murrell,
rejection of both N l brasiliensis and T l spiralis.
19 84)
the
MMC accumulation in
H l diminuta infected mice has been reported as beginning at the onset
of
rejection (Andreassen et al., 1978).
The use of mast cell deficient
in
W/WV mice to study worm rejection
the absence of mast cells has also produced various results. When
8
compared
to
mast
cell
normal
littermates, the
rejection
of
N.
brasiliensis from W/Wv mice has been reported as being both delayed
(Crowle and Reed, 1981) and normal (Uber et al., I98O).
Rejection of
I. spiralis from W/Wv mice has often been reported as being delayed (Ha
et al., 1983; Alizideh and Murrel, 1984; Kamiya et al., 1985).
Repair
of the mast cell defect with normal littermate bone marrow transplants
results in accelerated rejection of I l spiralis (Ha et al., 1983)
but
does not alter the rejection time of M. brasiliensis (Crowle, 1983).
These
conflicting results have led to a variety
of suggested
roles for MMC in the host response to intestinal parasites.
Various
investigators have suggested that MMC are involved in I) the rejection
of
the
parasite,
parasite,
and
3)
2)
regulation
repair
of
of
tissue
the
following
(Urquhart et al., 1965; Lewis and Austin,
1979) •
The suggestions have also
cellular
response to the
parasite
1981; Ferguson and Miller,
been made
that
mast
recruited by the parasite to benefit its establishment
(Miller and Jarret, 1971;
parasite
rejection
Mitchell, 1979),
cells
in the host
or are nonfunctional
rejection and occur only as part of an inflammatory
effect (Taliaferro and Sarles, 1939)•
are
in
side
In light of all observations and
suggestions, it is generally accepted that MMC play a functional, but
not essential, role in the host response
1979; Mitchell, Crowle and Reed,
to parasite invasion (Kay,
1981; Lee and Wakelin,
1982; Ellner
and Mahmoud, 1982; Michell et al., 1983; Alizideh and Murrell, 1984).
The complexity of the response, which involves the interaction of both
immunogenic and nonimmunogenic
components,
is not
disputed
(Befus,
9
1977; Kay, 1979; Mitchell, 1979; Askenase, 1980; Dessein et al., 198.1;
Castro, 1982; Ellrier and Mahmoud, 1982).
Functional activity of stimulated mast cells
Mast cells are stimulated by specific IgE to release a variety of
nonspecific
chemical
Specific IgE
is
mediators
produced
by
from
their
cytoplasmic
antigen-stimulated
granules.
B-Iymphocytes
in
mesenteric lymph nodes (MLN) (Ishizaka et al., 1976; Mayrohofer et al.,
1976).
IgE binds, with high affinity, to Fe receptors on the mast cell
surface (Ishizaka et al., 1972).
Subsequent cross-binding of IgE with
antigen initiates the release of granule
1973; Barret and Metcalf,
attributed
to
mast
cell
1984).
contents
(Jarret and Path,
Various biological activities
mediators,
which
are
integral
are
to
the
prospective MMC roles listed above.
Some
mediators
chemotactic factors,
are preformed,
such as histamine,
proteolytic enzymes,
and
serotonin,
proteoglycans,
while
others are newly syrithesizied upon stimulation, such as prostaglandins,
leukotrienes/ slow reacting substance
platelet
1984).
activating factor.
The
varied
of
anaphylaxis
(Wasserman,
biological
1979;
activities
of
Barret
(SRS-A), and
and Metcalf,
histamine
and
the
arachadonic acid metabolites include increased vascular permeability,
smooth muscle
transport
biological
contraction,
(Kelly
and alterations
and Dineen, 1976;
activities
may
of
transepithelial
Harari et al.,
contribute
to
parasite
1987).
ion
These
rejection
by
initiating changes in the intestinal environment which are detrimental
to the parasite.
Increased permeability of capillary endothelium may
enhance the passage of leukocytes and serum antibodies into the tissue,
10
thereby supplying the
intestinal
microenvironment
with
potential
parasite-damaging factors (Jones and Oglivie, 1971; Wasserman, 1979) •
Smooth muscle contraction, manifest as peristalis, may promote parasite
rejection by simple mechanical action
(Kelly and Dineen, 1976).
alteration in intestinal
ion transport
movement
the
of
fluid
into
intestinal
resulting
in
an
An
increased
lumen may promote parasite
expulsion both mechanically and physiologically (Castro, 1982; Harari
et al., 1987).
It is also possible that mast cell mediators, alone or
in conjunction with other mediators,
parasite
cause damage
directly
to
the
(Kelly and Dineen, 1976; Capron et al., 1978; Henderson et
al., 1980).
Mast
cells are the
source of several
factors chemotactic for
esoinophils collectively identified as eosinophil chemotactic factor of
anaphylaxis (ECF-A)
(Boswell et al., 1978; Wasserman, 1979)•
Two of
the factors have been isolated and identified as tetrapeptides with the
ability to selectively recruit and retain eosinophils at the site of
mast cell accumulation (Kay and Austen,
1971; Boswell et al., 1978).
Mast cells thus influence the influx of eosinophils which may in turn
influence
the
activity
of
h i s t a m i n a s e , arylsufatase
mast
and
cells
through
phopholipase
the
D which
release
of
inactivate
histamine, SRS-A and platelet activating factor, respectively (Boswell,
1978; Wasserman, 1979; Askenase, 1980).
MMC may perform a role in maintaining the homeostasis of the
intestinal microenvironment by regulating the activity of other cell
types.
For example, histamine released from mast cells may influence
T-Iymphocyte activity by binding histamine type 2 (H2) receptors on the
11
!-lymphocyte
surface
(Wassem a n , 1979;
Askenase, 198O;
Wasserman,
1984).
Since parasites usually cause some degree of damage to the host
tissue and MMC are often
observed
in
greatest
numbers
parasite rejection, MMC may also function in tissue repair.
released by MMC, designated rat mast cell protease II
active
on collagen type
following
A protease
(RMCP II), is
IV and may contribute to the
shedding and
reconstruction of the infection-altered intestinal epithelium (Woodbury
and Miller,
1982; Lewis and Austen,
rodent mast cells
1981).
Serotonin released from
may also be involved in repair by influencing
the
crypt renewal rate (Castro 1982).
An alternate explanation for the observations of greater numbers
of MMC subsequent to rather than at the time of parasite rejection is
based on the functional nature of the cell.
If the MMC responds to
parasite stimuli by degranulating, then it may be unstainable at the
time it is most active.
The observable mast cell response may occur
after the stimulus for degranulation is removed (i.e. the parasite) but
while
the
stimuli
for
development
of the cell
(i.e.
activated T
lymphocytes) are still present (Askenase, 1980).
Heterogeneity, fixation, and staining
The heterogeneity of the two mast
cytochemieal
in nature.
RMCPII
is
cell
populations is largely
associated with MMC, while a
chemically distinct RMCPI is associated with CTMC (Woodbury and Miller,
1982).
Histamine and serotonin are present at lower levels in MMC than
in CTMC as demonstrated by. fluorescent staining
(Miller and Walshaw,
1972; Askenase,
1984).
1980; Saavedro-Delgadra et al.,
MMC granules
12
contain a proteoglycan
(glycosaminoglycan or GAG) which is less highly
sulfated than the GAG, heparin, in CTMC (Miller and Walshaw, 1972; Tas
and Berndsen, 1977)•
The GAGs of both CTMC and MMC
serve primarily as
structural matrices in the granules, binding other granule components
by virtue of their high negative charge (Miller and Walshaw, 1972; Tas
and Berndsen, 1977).
The less sulfated GAG in MMC granules provides
fewer anionic sites for the binding of cationic dyes and may contribute
significantly to the special staining and fixation requirements of MMC
(Enerback, 1966a; Enerback, 1966b; Miller
and
Walshaw,
Mayrohofer, 1980; Crowle and Phillips, 1983; Crowle, 1985).
1 972;
Fixatives,
like Carnoy1s, containing a mixture of alchohol and acetic acid are
effective preservatives for MMC (Smirzai, 1963; Enerback 1966a; Crowle
and Phillips, 1983).
unless
it
Formalin preserves CTMC but does not preserve MMC
is modified with
Crowle and Phillips, 1983).
acetic acid
(Miller and Walshaw,
Binding of cationic
1972;
(basic) dyes to the
anionic GAG causes the characteristic metachromasia of stained mast
cell granules
(Smirzai,
1963)•
Effective basic dyes include
astra
blue, alician blue and toluidine blue of which astra blue is often most
effective (Enerback, 1966b; Crowle and Phillips, 1983; Crowle, 19,85).
Staining
and
fixation
differences in CTMC and MMC may
also
reflect different structural charactersitics of the two subtypes.
The
granules of MMC are characteristically smaller,
more variable in shape than those
fewer in number and
of CTMC. (Murray
Enerback and Lundin, 1974; Crowle and Phillips, 1983).
et
a l .,
1968;
Paracrystalline
structures observed in rodent MMC increase with parasite infection and
may represent a remnant matrix following loss of granule components or
13
a matrix in the process of acquiring newly generated material (Murray
et
al.,
1984).
1968;
Crowle
and Phillips,
1983;
Handlinger and Rothwell,
The secretion of granule contents may occur by different modes
for CTMC and MMC.
CTMC can be induced to degranulate by several agents
which have no effect on MMC.
venom peptide 401
This includes the secretgoque 48/80, bee
(Enerback and Lundin,
1974;
Pearce et al.,
1982;
Barret and Metcalf, 1984), various neuroenteric peptides (Shanahan et
al., 1985) and endorphorins (Shanahan et al., 1984).
micrograph (EM) observations,
through
1965;
CTMC release granule components either
exocytosis or through release
Murray et al.,
1968;
Based on electron
of the entire granule
Barret and Metcalf,
1985).
(Mann,
Release
of
granule contents from MMC may occur with complete lysis of the cell or
may involve migration of the cell to the tip of the intestinal villus
and discharge of contents directly into the intestinal lumen (Murray et
al., 1968; Miller, 1971; Crowle and Phillips, 1983).
Any cell remnants
may be subsequently phagocytosed by eosinophils or macrophages (Mann,
1965; Miller and Jarret, 1971)*
Origin, differentiation, and development
A common bone marrow origin of MMC and CTMC was established
through
transplantation
experiments.
Irradiated C57BL mice were
injected with various lymphoid and hematopoietic tissues from congeneic
C57BL beige (bg/bg) mice whose genetically defective granulocytes have
enlarged granules which
transplanted tissue.
serve as indicators of the donor source of
Transplants of bone marrow cells,
but not of
thymus, MLN, or Payer's patch cells replenished the CTMC population in
the skin of recipient mice.
The same results were achieved using mast
14
cell deficient W/Wv mice as recipients of normal littermate tissues
(Kitamuraf 1979b)..
MMC were
shown to derive
from
tissues through examination of the,MMC response
infected
W/Wv
mice
which
had
hematopoietic and lymphopoietic
received
tissues
hematopoietic
to Ni brasiliensis
transplants
from normal
of
various
littermates.
Transplantations of bone marrow and spleen, but not of thymus tissue
replenished the MMC population in the intestinal mucosa of infected
recipient mice (Crowle and Reed, 1984).
These two studies further helped establish thymus independency of
the CTMC and thymus dependency of the MMC through
the incorporation of
athymic nude mice into their experimental protocols.
skin grafted onto the back of athymic nude mice
number of mast
(Kitamura,
cells,
1979b).
developed a normal
demonstrating the thymus independence of CTMC
The MMC response was restored in nude mice
thymus transplantation,
lacking functional
A piece of W/Wv
but not
thymocytes,
by
the
transplantation
such as. bone marrow,
thymus dependency of MMC (Crowle and Reed, 1981).
vitro and in vivo studies,
tissue
demonstrating a
Based on both in
the thymus dependency of MMC is strongly
believed to be a regulatory
activated T lymphocytes
of
by
lymphokine released
from
specifically
(Ruitenberg and Elgersma, 1976; Mayrohofer,
1979; Ginsberg et al., 1981; Haig et al., 1982; Reed et al., 1982).
CTMC and MMC have been shown to arise in the bone marrow from
the same
stem cell
Greenberger et al.,
(Kitamura et al., 1981;
1983;
Sharkis
Suda et al., 1985).
commitment to a specific mast
cell
precursor
et
al.,
The level
occurs
is
1981;
at which
unknown.
Granulated mast cells do not exist in the bone marrow or blood (Crapper
15
and Schrader, 1983;
states,
such
Guy-Grand, 1984)
as mast
migration of mast
cell
show that mast
leukemia
cells, through
demonstrated•by Kitamura
except
in mast
cell
(Travis et al.,
disease
1986).
The
the blood to the tissue was first
(1979a) who used beige-normal parabionts to
cells with both enlarged and normal
developed equally in each recipient.
sized
granules
Circulating precursor mast cells
undergo some differentiation prior to localization in the tissue since
undifferentiated stem cells can not be demonstrated in gut tissue by
CFUs (colony forming units-spleen; macroscopic spleen colony assay for
non-differentiated stem cells) (Guy-Grand, 1984).
The final maturation
into granulated cells and differentiation into heterogenous subtypes is
believed to occur under a local
tissue influence
(Wlodarski,
1976;
Kitamura, 1979c; Pitts and Mayrhofer, 1983; Haig et al., 1984; Nakano
et al., 1985).
Local differentiation of mast cells into specific subtypes under
influences
of
the
local
tissue
enviro n m e n t
was
impressively
demonstrated through the application of cultured mast cells.
Nakano et
al. (1985) injected W/Wv mice with either cultured mast cells or CTMC
from the peritoneal cavity of normal mice, then
examined the tissues
of recipient mice and found that regardless of the source of donor
cells, mature mast cells developed in both the connective tissue and
mucosal tissue of recipient mice.
in
recipient
W/Wv mice
had
the
Further, mast cells which developed
morphological
and
histochemical
characteristics of CTMC when located in the connective tissues and of
MMC when located in the mucosa.
16
In vitro cultivation of mast cells has contributed greatly to the
understanding of this cell.
of tissues,
Mast cells can be cultured from a variety
including bone marrow, thymus, spleen,
lymph nodes,
and
intestinal tissue (Ginsberg et al., 1981; Haig et al., 1982; Crapper
and Schrader, 1983; Guy-Grand, 1984).
the morphological
including
and histochemical
dependency
on
a growth
Cultured mast cells have many of
properties of the MMC
subtype,
factor derived from activated T
lymphocytes, most commonly referred to as interleukin 3 (IL3) (Haig et
al., 1982;
Greenberger et al., 1983;
cultured mast
cells by other
evidence
local
for
subtypes.
Guy-Grand,
1984).
study groups has provided
differentiation
of mast
cells
The use of
additional
into
specific
Guy-Grand (1984) showed that mast cells cultured from the
intestinal tissue are more highly differentiated than mast cells grown
from other tissues based on differences in cell surface markers and
secretory properties.
At least two groups of investigators (Tertian et
al., 1981; Schrader et al., 1983) have shown that mast cells can be
cultured from the intestines of both normal and nude mice, indicating
that the influence of the !-lymphocyte on differentiation of the mast
cell precursor into the MMC occurs in the intestine.
Local
proliferation
substantiated
by
the
and
clonal
maturation
nature
of
of
the
mast
cell
proliferating mast
is
cells
(Kitamura, 1979c), the presence of mitoses in proliferating populations
(Miller and Jarret, 1971), and to some extent by electron and light
microscopic observations of the developing mast
Combs, 1971; Miller and Jarret, 1971).
cell
(Murray, 1968;
The immediate precursor mast
cell is described as lymphoid, but a direct relationship between the
17
lymphoid
cell and the mature
mast
cell, which
has
metachromatic
staining granules and IgE surface receptors, has not been established
(Murray, 1986; Miller and Jarret, 1971).
It has been suggested (Guy-Grand,
1984) that MMC derive from a
population of cells referred to. as intraepithelial leukocytes
(IEL),
which share morphologic and histochemical properties with mast cells,
such
as
metachromatic ally-staining
histamine.
granules
and
intra c e l l u l a r
A clearer relationship has been established between IEL and
natural killer (NK) cells based on common cell surface markers and in
vitro growth properties (Ernst et al., 1985)•
the intestine
The IEL "compartment" of
includes a variety of cell types some of which may be
involved in mucosal protection (Ernst et al., 1985).
The relationship between MMC and another intraepithelial cell,
the globular leukocyte,
has been more
clearly
defined.
leukocytes share many of the characters!tics of mast
addition of large acidophilic granules
cells with the
(Murray, 1968).
were thought to be independent of mast
Globular
These cells
cells located in the lamina
propria by at least one study group (Ruitenberg and Elgersma, 1979).
They are considered by many investigators to be partially degrEmulated
end-stage mast cells which have migrated from the lamina propria to the
intestinal epithelium (Murray et al., 1968; Miller and Jarret, 1971;
Mayrohofer, 1980;
Huntley et al., 1984),
and are
often
considered
together with lamina propria mast cells, as a single MMC population.
Mast cells both emigrate from the.bone marrow and proliferate
locally under the appropriate stimuli (Keller et al., 1974).
The exact
nature of the stimuli which initiate the MMC response is unknown, but
18
assumed to be related in some manner to the parasite
1971; Miller and Jarret, 1971; Nawa and Miller,
Products released from the parasite
metabolic)
These
1979; Castro,
excretory,
1982).
secretory,
are likely processed as antigens at the mucosal surface.
products may
through
(somatic,
(Murray et .al.
the
stimulate the mast
activation
cell directly
or
indirectly
of other components of the response.
The
distribution of MMC throughout the intestine during parasite infection,
rather than just at the site of burden (Keller, 1971; MacDonald et al.,
1980)
and at
sites experimentally detached from
(Pitts and Mayrohofer, 1983)
activation of mast cells.
gut-homing
behavior
parasite
exposure
refutes the concept of direct parasite
Antigen stimulated T lymphocytes, which show
(Rose et al.,
1978)
and are essential
for MMC
differentiation, could release numerous lymphokines to coordinate such
T-cell
dependent
functions as proliferation of MMC
Elgersma, 1976) production of specific IgE antibody
(Ruitenberg and
(Isizaka et al.,
1976; Reed et al., 1982), and enhancement of eosinophil accumulation
(Basten and Beeson, 1970; Hindsbo et al., 1982).
Parasites may
release
specific factors which
induce mast
cell
activity to benefit their own establishment in a compromised intestinal
wall (Miller and Jarret, 1971).
then be the
physical
This early induced degranulation would
stimulus for subsequent mast
presence
of
cell
accumulation.
The
the parasite has also been suggested as the
stimulus for the MMC response.
If the MMC is primarily a repair cell
or is recruited as part of an inflammatory side effect, then mechanical
irritation of the mucosal surface may explain the influx of these cells
(Taliaferro and Sarles, 1939; Nawa and Miller, 1979; Askenase, 1980).
19
Interaction with IgE
The
synchronous
development
of
parasite
specif c
IgE
by
antigenically stimulated B-blasts in the mesenteric lymph nodes (MLN)
would facilitate IgE dependent mast cell degranulation and may provide
an additional stimulus for mast
cell growth (Mayrhofer, 1976).
The
association of mast cells with IgE in the intestines of parasitized
animals
has
been
demonstrated
(Mayrhofer et al., 1976;
with
immunofluorescence
Alizadeh et al., 1984),
and
techniques
it has
been
clearly established that mast cells serve as the main repository for
IgE in the tissues (Mayrohofer, 1976; Jarret and Path, 1973; Dessein et
al., 1981; Kaliner, 1979).
The binding of IgE to MMC could occur in two possible locations,
at the mucosal surface or in the MLN.
Binding of IgE by mast cells in
the mucosa would occur after IgE, produced in the MLN, had circulated
unbound in the blood and lymph to the mucosa (Mayrhofer et al., 1976).
Binding in the MLN would likely involve a precursor mast cell, which
would enter the circulation with bound IgE and return, or "home", to
the intestinal mucosa
explain
why
serum
characteristically
(Gillion, 1981).
IgE,
normally
The latter possibility would
occurring
in
elevated later in infection,
low
but
levels,
is
is not evident
prior to MMC accumulation (Oglivie, 1964; Jarret and Path, 1973; Jarret
et al., 1976; Dessein et al., 1981; Ishida and Yoshimura, 1986).
would also explain the distribution of MMC throughout
This
the intestine
rather than just at the site of parasite burden (Gillion, 1981).
20
Interaction with eosinophils
The MMC response is often accompanied by eosinophilia
1962;
Mann,
1965;
Kelly
and Oglivie, 1972;
Oglivie
et al., 197.7;
Ruitenberg et al., 1977; Kay, 1979; Dessein et al., 1981).
and MMC may interact synergistically at several levels.
(Wells,
Eosinophils
ECF-A released
from MMC granules attracts, retains and may also enhance the function
of eosinophils.
(Capron et al., 1978; Kay, 1979; David et al., 1980).
Eosinophils
adhere
to
the
surface
shistosomula (Capron et al., 1978) and
presence of specific antisera.
of
Schistosoma
mansoni
brasiliensis in vitro in the
In the case of
mansoni adherance is
accompanied by damage to the schistosomula (Butterworth et al., 1975;
Capron
et
al.,
1978,
capacity of eosinophils
David et al.,
1980).
The parasite-damaging
is attributed to two major components of the
eosinophil
granule, the major
peroxidase
(EPO)
basic protein
(Kay, 1979).
The
basic
(MBP)
nature
and eosinophil
of
eosinophil
components facilitates interaction with the acidic GAG of the mast cell
granule resulting in a
complex which may have
greater
cytotoxic
potential than the eosinophil alone (Henderson et al;, 1980).
Eosinophils function both dependentIy and independently from T
lymphocyte regulation (Goven, 1983)•
Eosinophilia has been observed in
athymic nude mice in response to some parasites
(Andreassen et al.,
1978; Ishida and Yoshimura, 1986) but is absent in response to others
(Ruitenberg et al., 1977; Sugane and Oshima, 1982).
This may. reflect
the extent of the interaction of the parasite with the host tissue.
21
Parasite and host influence
Parasites with larval stages that migrate through the tissues and
with adult stages that exist in intimate contact with the intestinal
villi, such as
1982), might
spiralis and Ni brasiliensis (Wright, 1979; Kassai,
elicit a stronger
host
response
than a noninvasive
cestode, such as Hi diminuta. which has limited direct contact with the
intestinal
mucosa of the host
1979; Ha et al., 1983).
through
its scolex
(Gray,
1976; Kay,
However, the MMC response has been shown to
occur even when the larval stage is eliminated and when the parasite is
removed from direct contact with an experimentally isolated intestine
(Pitts
and
Mayrohofer, 1983).
Furthermore,
functional
released from parts of the parasite not in direct
intestine,
such
as the
tapeworm
antigens
contact with
the
tegument, may contribute to the
stimulation of the host response (Befus, 1977).
Inherent
species,
strain,
differences in the parasite host,
and individual animal,
degree of the host
1982).
response
at the levels of
also affects
(Read and Voge, 1954;
the nature and
Lee and Wakelin,
The comparison of Hi diminuta infections in rats, and mice is an
example of a species difference in the response to a parasite.
Normal
mice reject Hi diminuta within 10 days post-inoculation while normal
rats retain the parasite for
several
months
(Read and Voge, 1954).
This difference in host response to the Hi diminuta is attributed to
species variation of the intestinal microenvironment and immune system
(Hopkins et al., 1972).
The use of one animal
variations,
strains of the same species also exhibit
but different
species limits the
variability in their responses to a parasite (Lee and Wakelin, 1982).
22
Mice experimentally bred to be deficient in a given component of the
response, such
as
the MMC, provide effective tools for controlled
examination of the host protective mechanism.
Animal models
The mast
cell
defect
of W/Wv
mice
was
first
recognized by
Kitamura (1978) who reported that W/Wv mice had in their skin less than
1% of the CTMC than the levels found in normal littermates.
The mast
cell defect was subsequently found to include MMC (Uber et al., 1980;
Crowle
and Reed, 1981).
W/Wv mice are bred as the, Fl generation of
WBReJ-W/+ x C57BL/6J-Wv/+.
and Wv /+
are
fertile,
(Russell,
1979).
Littermate mice with genotypes +/+, W/+',
black or
spotted and hematologicalIy normal
Mice with genotype WZWv are sterile,
solid white,
affected with macrocytic anemia and have deficiencies of granulocyte
populations to varying degrees (Chervenick and Boggs, 1969)•
The WZWv
abnormalities are attributed to a hematopoietic
related
stem-cell
defect based on the observation that WZWv bone marrow cells produce
limited .CFU-s
irradiated
(macroscopic
littermate
mice
spleen
colonies)
(McCulloch, 1964;
when
injected
Lewis,
1967).
into
The
lymphopoietic stem cells of WZWv mice appear to be largely unaffected
since major functions associated with B and T lymphocytes are normal
(Mekori and Phillips,
1969; Reed et al., 1982; Thomas and Schrader,
1983; Ha et al., 1986).
WZWv mice do, however, accept transplants of
parental bone marrow, without developing a graft vs. host reaction even
though the donor
(Sharkis , 1981;
cells populate the recipient hematopoietic tissues
Harrison,
1976), which
indicates
some
immunologic
23
abnormality in the donor lymphocytes, the recipient lymphocytes, or the
interaction between donor and recipient lymphocytes (Harrison, 1976).
/
The possibility of a T lymphocyte related defect in W/Wv mice is
supported to
some degree
competency tests.
(1979b)
by transplantation experiments
and
immune
Wikor-Jedzejzak et al. (1977) and Kitamura et al.
injected W/Wv mice with
anti Thy-I
treated bone marrow and
noted a repair of the CFU-s producing capacity and of the CTMC defect,
respectively, but not of the anemia.
Through a series of experiments,
Wikor-Jedzejzak et al. (1977), concluded that a.thymus regulating cell,
they termed anti-theta sensitive regulatory cell
deficient or altered in W/Wv mice.
(anti-TSRC), may be
Mekori and Phillips (1969) repaired
irradiated animals with different combinations of normal and W/Wv bone
marrow and thymus cells.
They determined that the thymus and bone
marrow cells of W/WV mice adequately repaired the immune competency of
SRBC-challenged recipient mice,
using the plaque forming cell
(PFC)
assay, but demonstrated a greatly increased synergism in PFC producing
ability
when
the
repair
cells
consisted of W/Wv bone marrow and
littermate thymus.
Most lines of evidence suggest that the "stem-cell related" mast
cell
defect
of
the W/Wv resides in the tissue
animal, rather than in stem cell compartment.
cultured
from
the
bone
marrow,
blood,
and
compartment of the
Mast cells have been
spleens
of W/Wv
littermate mice in similar numbers, but in far fewer numbers
intestines
1983).
of W/Wv mice
(Suda et al.,
1985;
and
from the
Crapper and Schrader,
All observations of mast cell deficiency indicate that the cell
is missing or is non-functional in tissues (McCulloch, 1964; Kitamura
24
et al., 1978).
The fact that repair of the W/Wv defect with normal
littermate bone marrow always involves repopulation with the donor type
cells
(Seller, 1968:
Harrison
et
al.,
1979)
indicates that
some
regulatory factor may be transplanted along with the stem cells in the
donor bone marrow which contributes to W/Wv repair.
factor" may be T cell related,
or may be another
This "regulatory
component
which
locates and influences the development of cells within tissues.
The absence of functional
permanent replacement
(Russell.et al,
1959;
mast
cells in W/Wv mice,
by injection of normal
Crowle and Reed,
littermate
and their
bone marrow
1984) provides a valuable in
vivo model for the investigation of the role of the mast cell against a
specific
stimulus
(Kitamura
et al., 1985), such as an intestinal
parasite. The functional capacity of the W/Wv immune system has been
shown to be comparable
to
that
of
normal
mice
in
both B and
T
lymphocyte functions, such as specific antibody production and delayed
type hypersensitivity
(DTH)
responses
(Mekori
Sharkis et al., 1981, Ha et al., 1986).
and
Phillips,
1969;
Reports of MMC occurring in
the intestinal mucosa of infected W/Wv mice have varied from none (Uber
et al., 1980; Crowle, 1983; Crowle and Reed, 1984) to a very few (e.g.
3 MMC/10 VCU in W/Wv vs.
537 MMC/10 VCU in LM) (Ha et al., 1983;
Mitchell et al., 1983; Alzideh and Murrell, 1984; Kamiya et al., 1985).
The athymic nude mouse is essentially hairless and lacks a normal
thymus
(Flanagan,
1966; Pantelouris, 1968).
It is commonly bred by
crossing nu/nu males with nu/+ females since the nu/nu female can be
less fertile
(Amstutz
and are less successful
et al., 1976).
nurturers of
their
offspring
The nude gene has been bred onto several
25
genetic backgrounds, such as C57BL and BALB/c,
with elected congenic strains (Guide, 1976).
which allows, studies
A thymus remnant occurs
in nude mice which consists of bands or lobes of vesiculated tissue
containing
abnormal
"epithelial-like"
(Pantelouris and Hair,
1970;
cells
and
Owen et al., 1975) •
no
lymphocytes
The thymus in a
normal animal consists of a lymphoid component which is derived from
precursor cells migrating from the fetal liver and bone marrow, and an
epithelial component which "programs" or "educates" the precursor cells
to become functional T lymphocytes (Owen et al., 1975).
defect
lies
in
the
The nude mouse
defective development of the thymic epithelium
(Wortis, 1971; Guide, 1976).
A precursor T cell (T-lineage, T-Iike)
has been demonstrated to exist in the bone marrow, spleen and lymph
nodes of nude mice by fluorescence localization and Thy-I cell surface
labelling (MacDonald et al., 1981; Ropke,1977).
differs
from
the
functional
T
lymphocyte
in
The
T-lineage cell
that
it
does
not
recirculate through the thoracic duct, has a shorter lifespan, and has
a lower density of the Thy-I surface marker (Roelants et al., 1976;
Amstutz et al., 1976).
The T-lineage cells present in nude mice may
have a limited capacity to function as normal T lymphocytes (MacDonald,
1984).
The nude mouse mutation is manifest as a deficiency in functional
T lymphocytes.
responses,
Nude
mice have
severely
such as graft rejection,
antibodies against many antigens
a l . , 1970) .
(Bamberger et
Nude
mice
al., 1977).
are
impaired
cell
mediated
and lack the ability to produce
(T-dependent antigens)
additionally
anemic
(Manning
et
and
leukopenic
Littermate heterozygotes are
apparently
26
hematologicalIy normal and have normal T lymphocyte activities (Guide,
1976).
Myeloid
(Bamberger,
precursor
1977).
cells occur in normal
levels in nude mice
Mast cells can be cultured in conditioned media
from the the bone marrow, spleen, and intestine of nude mice (Tertian
et al., 1981; Schrader et al., 1983).
Nude mice contain normal
higher than normal numbers of CTMC (Wlodarski, 1976).
to
MMC are absent
or present in low numbers in the intestines of nude mice (Crowle, 1983;
Crowle and Reed,
al.,
1978).
1981;
Other
Ruitenberg and Elgersma, 1976; Andreassen et
cells
under
thymic
influence, such
as
the
eosinophil, have been reported to be both absent (Ruitenberg et al.;
1977; Goven, 1982) and present at normal or higher than normal levels
in nude mice
(Andreassen et al.,
1978;
Ishida and Yoshimura, 1986;
Sugane and Oshima, 1982).
The host
response
to parasites is complex.
It involves the
interaction of several components of the host protective system and is
dependent
upon both the status of the host and the nature of the
parasite.
Animals bred to be specifically deficient in a component of
the host protective system, as are mast cell deficient W/Wv mice and
thymus deficient nude mice, are useful models for evaluating the role
of specific protective components on the capacity of the host to deal
with a specific parasite stimulus.
27
METHODS AND MATERIALS
Mice
WBB6FI-WZWv (WZWv) mice were obtained by crossing WBReJ-WZ+ and
C57BLZ6J-Wv Z+ mice.
Littermate (LM) mice of genotypes WZ+, WvZ+, and
+Z+ were used as control mice.
Mice used experimentally were between
4-8 months old and of both sexes.
C57BLZ6J-nuZnu
(C57BL
nude) mice
were
obtained by crossing
C57BLZ6J-nuZ+ females with C57BLZ6J-nuZnu males.
C57BL nude mice used
as control in the WZWv (Tenebrio molitae) experiment were between 5-10
months old and all male.
These mice were considered separately from
the C57BL mice used in the nude mouse experiment.
in the nude mouse experiment were
sexes.
C57BL nude mice used
between 2-4 months old and
Littermates to C57BL nude mice
both
(C57BL LM) were between 2-4
months old, both sexes, and were either heterozygotes or homozygotes.
BALBZc-nuZnu (BALBZc nude) mice were obtained by crossing BALBZcnuZ+ females with BALBZc-nuZnu males.
Littermate mice (BALBZc LM) were
either heterozygotes or homozygotes.
BALBZc mice used experimentally
were between 2-4 months old and of both sexes.
All mice used in this study were bred at the Animal Resource
Center,
Montana State. University,
Bozeman,
MT
from
breeding
stock
orginally obtained from Jackson Laboratory, Bar Harbor, ME.
Bone marrow repair
WZWv
bone
marrow
repaired mice
(WZWvBM)
received bone marrow
transplants from sex and age matched normal littermates.
Bone marrow
28
cells were aseptically removed
from
the
femurs
of
donor
animals,
checked.for viability by Trypan Blue exclusion, and resuspended to 4 x
107 bone marrow cells per ml
(Mishell and Shiigi,
1980).
Recipient
mice were injected intravenously (IV) in the lateral tail vein with 0.5
ml of prepared cells (2 x 10? cells/ mouse) (Crowle, 1983; Kitamura et
al.,
1979)«
WZWvBM mice
were
allowed
to
normalize
(Kitamura et al., 1979) before being used experimentally.
for 60 days
Repair of
the anemia and mast cell defect were checked by hematocrit and by the
presence of CTMC in the skin of the ear.
Mice with hematocrits of 47-
53% were considered to be successfully repaired (WZWvBM)
al., 1981).
(Sharkis et
WZWv mice had hematocrits of 34-44% and a control group of
littermate mice had hematocrits of 48-58%.
CTMC in the ears of all
mice were stained by the method described below for MMC.
Parasite
H. diminuta cysticercoids were recovered from the intermediate
host flour beetle, T^ molitae (Carolina Biological, Burlington, N O
the
intermediate
Denmark).
reported
host
flour beetle,
T l cdnfusum
or
(Jorn Andreassen,
Experiments using each source of infective cysticercoid were
separately
and
compared for
significant
differences.
T.
molitae was the intermediate host source for H l diminuta in all cases
where the intermediate host is not specifically indicated.
Cysticercoids were dissected from beetles into Hanks' Balanced
Salt Solution (HBSS)
(Irving Scientific, Santa Rosa, CA).
Mice under
light ether anathesia were injected via gavage with 3 cysticercoids per
mouse (Andreassen and Hopkins, 1980; Isaak, 1983).
The gavage tube was
checked under a dissection scope (30x) after each injection to insure
29
that all 3 cysticercoids were administered to each animal (Hesselberg
and Andreassen, 1975).
Day 0 data were derived from animals which
received no cysticercoids.
Experimental protocal
Mice in all experiments received 3 cysticercoids of
except those used for day 0 data.
diminuta,
Three-worm injections were used to
-provide adequate stimulus for a histologically detectable host response
and for the recovery of worms of manageable number and size (Befus,
1975; Andreassen, personal communication).
Seven groups of mice were
used experimentally:
GROUP
ABBREV.
THYMUS
CTMC
MMC
Littermate
LM
+
+
+
W/Wv
WZWv
+
-
-
repaired
W/Wv bone marrow ;
WZWvBM
+
+
+
C57BL nude
C57BL NU
-
+
-
C57BL littermate
CS7BL LM
+
+
+
BALb/c nude
BALB/c NU
-
+
.-
BALB/c littermate
BALB/c LM
+
+
+
Littermate, W/Wv , WZWv bone marrow repaired mice
Intermediate host: Tenebrio molitae
On each of days 0, 6/7 , 10, 12, 16, and 21 post-inoculation
5-10 LM and W/Wv mice were killed; on day 24 PI, 3 LM and 2 WZWv
were killed; five WZWvBM mice were killed on each of days 7 , 12,
16, and 21 PI; and two C57BL nude mice were killed on each of days 7,
30
10, 16, and 21 PI.
All mice were randomly selected for sacrifice
except those specifically non-infected for day 0 data. .
Littermate mice were used as the normal mouse model.
were used as a .model
repaired
model.
W/Wv mice
of total mast
Bone marrow
were used as a specifically mast
C57BL nude mice
deficiences,
cell deficiency.
were
used
as
including the lack of MMC.
infection positive controls,
extended periods of time
since
a model
of
W/Wv mice
cell-restored
thymic-related
Nude mice were also used as
I) they retain Hi diminuta
(Isaak et al., 1975)
and,
for
2) Hi diminuta
becomes established in mice but does not always produce eggs (Hindsbo
et al., 1982), which limited the use of a standard eggs per gram feces
(EPG) assay for positive infections.
Littermate and W/Wv mice
Intermediate host: Triboliun confusun
Five LM and five W/Wv mice were killed on each of days 6 , 12, 18,
and 24 PI.
The response of the
host
to worms
derived
from
the
intermediate host T i confusum in this experiment was compared to the
response of the host to worms derived from the intermediate host Ti
molitae.
Nude mice
Three mice from each group were sacrificed on each of days 0, 7,
12, 16, and 21 PI.
C57BL nude mice were compared to the more commonly
used BALB/c nude mice for their response to H i diminuta. Littermate
mice of both strains were included as controls.
31
Worm recovery
Mice were killed by cervical
dislocation on various days PI.
Small intestines were removed and flushed with saline to remove worms
and debris (Hopkins et al., 1972; Isaak, 1983).
On the earliest PI
days, both the collected debris and the slit intestine (prepared for
histology) were carefully examined under a dissecting scope (30x) for
small worms.
Further examination of the intestine
for small worms
through mucosal scraping or extended incubation (Hopkins et al., 1972;
Andreassen and Hopkins, 1980;
Hindsbo et al., 1982)
was omitted in
preference to maintaining the integrity of the intestinal wall
for
histological observations.
Under these
small worm;
circumstances, it was difficult
therefore,
earliest days PI
to recover every
the number of worms recorded as surviving on
(days 6 and 7) was considered to be a minimal value
(Hopkins et al., 1972; Andreassen et al., 1978).
does establish in mice
Since tL_ diminuta
(Andreassen et al., 1978, see also results in
this document) and the inoculation of cysticercoids was ascertained by
checking the inoculation tube, inoculated mice were considered to be
infection positive on days 6 and 7 PI in spite of the absence
detectable worms in some cases.
The
%
of
positive values represent the
number of animals positive for infection in each group.
The number of worms recovered was
based
upon
the
number
of
scolices with attached neck and proglottids, when whole worms were not
found (Andreassen et al., 1978).
recovered.
The
%
Iamost
cases, complete worms were
recovery values were determined by dividing the
number of worms recovered from a group of animals by the number
of
32
animals in the group x the number of cysticerooids administered per
animal.
Worm measurement
Recovered worms were placed in deionized water and kept at 4° C
overnight to relax, musculature and facilitate measurement
et al., 1978).
glass
under
The length of relaxed worms was measured on a piece of
a
dissection
scope
(15x)
with
a millimeter
(Hesselberg and Andreassen, 1975; Hindsbo et al., 1982).
were recorded in centimeters
weight
(Andreassen
of worms,
(cm) to the nearest 0.1
in milligrams
scale
Measurements
cm.
The dry
(mg), was determined after removing
surface liquid with filter paper and drying the worms overnight at 60°
C in preweighed tin foil cups (Hesselberg and Andreassen, 1975).
Worms
weighing <1.0 mg were considered to be 0.5 mg (Befus, 1975).
Worm length and weight values are given as the mean length and
weight values from all worm positive mice in a group on a given day PI.
Worm negative mice are represented on Length and Weight figures only
when all mice in a group were worm negative
(0% positive)
in which
cases the representative group symbol was placed on the origin line of
the x-axis.
Length and weight values for worm negative mice were not
included with values for worn positive mice when both occurred since
these
averages artifically
lowered the length and weight values of
recovered worms.
The lengths and weights of individual worms recovered from each
mouse were combined into total worm lengths and weights per mouse, i.e.
the measurements for each individual worm (1-3 per mouse) were added
33
together and considered as a single worm value per animal (Hindsbo et
al., 1982).
Histology
Each saline-rinsed intestine was stretched on a corkboard next to
a marked rubber band which could be stretched to the individual length
of each intestine
(Andreassen et al., 1978).
The
rubberband
was
divided into 5 sections, (I) 0-17$, (2) 17-30$, (3) 30-43$, (4) 43-56$,
and (5) 56-100$.
Section I of corresponding intestine was primarily
duodenum, section 2-4 jejunum, and section 5 ileum (Andreasen et al.,
1978).
Since most worms have been found in sections 2 and 3 of the
jejunal intestine (Andreassen et al., 1978), these sections along with
sections
I and
4 were
kept
for
histology.
Each
intestine was
transferred to a moistened strip of paper, slit longitudinally and
flattened XCrowle and Reed,
1981),
then cut
section was further divided in half.
into
sections.
Each
One half was placed in Carnoy1S
fixative (60$ EtOH, 30$ chloroform, 10$ glacial acetic acid) (Manual of
Histologic and Special Staining Techniques, 1949) for 3-8 hours for the
staining observation of MMC (Enerback, 1966a; Crowle, 1985), the other
half was placed in 10$ buffered formalin for the staining observation
of tissue eosinophils
Techniques,
(Manual
of
Histologic
1949; Andreassen et al., 1978).
and
Special
Staining
Tissues were dehydrated
and blocked in paraffin, then cut in IOum sections with the intestinal
villi in a longitudinal orientation.
Carnoy's fixed sections were stained in 0.5 astra blue at pH 0.20.3
(0.5 gm astra blue FM, Schmid GMBH and Co., in
100ml distilled
34
water with 6 ml 37% HCL)
washed in hematoxylin and eosin bluing agent
(0.2 gm NaHCOg and 2.0 gm MgSOjt . THgO in 100 ml of distilled water),
and counterstained with
0.25%
eosin
Y
(0.25
gm
eosin
Y , Fisher
Scientific Co., Seattle, WA, in 20 ml of distilled water and 80 ml of
100% ethanol). MMC stain a bright blue against a pink background with
this technique (Crowle, 1985).
Formalin
fixed sections were
stained in 0.5
Orange-G (0.5 gm eosin Y,Fischer Scientific
H a r l e c o , Philidelphia, in
100
ml
% Eosin Y /0.5%
Co., 0.5 gm
distilled
Orange
G,
wa t e r ) , washed
in
hematoxylin and eosin bluing agent (0.2 gm NaHCOg and 2.0 gm MgSOlt .
THgO in 100 ml of distilled water), and counterstained in Hematoxylin
(Gill II).
Tissue eosinophils
blue-violet
background with
stain a bright red-orange against a
this technique
(Litt,
1969; Manual
of
. Cells were counted and recorded per 10 villus crypt units.
A
Histologic and Special Staining Techniques, 1949)•
villus crypt unit
(VCU) is the area of the small
gland crypts and the villus above
intestinal mucosa
between
two
(Miller and Jarret,
1971).
Counts of intraepithelial mast cells and lamina propria mast
cells were combined and treated as a single population of cells (Crowle
and Reed, 1981).
Intestinal cell counts for a given day are means of counts from
all mice in a group killed on
negative.
that day PI, both worm positive and worm
In some cases, the number of animals included in the mean
cell count was less than the total number of animals killed due to the
unsuccessful histological preparation of some samples.
35Blood cell counts
Blood was obtained from the same littermate, W/Wv , and WZWvBM
mice on alternate days PI beginning on day -I and through day 21 PI.
Blood
was obtained from C57BL and BALB/c mice on alternate days PI
begining on day 0 and through day 20 PI. C57BL control mice in the WZWv
mouse experiment were sampled the day before sacrifice.
Blood samples
were taken during the same two hour period each day in an effort to
standardize the count since peripheral blood eosinophils levels have
been reported to have a diurnal variation (Colley, 1974).
Free flowing
blood from the lateral tail vein was drawn up into a standard white
blood cell pipette and mixed with Discombes dilutant
Eosin I,
0.5 ml
acetone, 10 ml distilled HgO)
(Basten et al., 1979)•
large
(0.5 ml aqueous
made fresh each day
Red-staining eosinophils were counted in four
squares of a Neubauer bright line hemacytometer chamber,
and
reported as eos/mmS of blood using the following formula (Mishell and
Shiigi, 1980):
cells/mm^ = (avg.
4
cells in 4 large squares)(10)(1/dilution)
dilution (standard white blood cell pipette) = 1/20
The peripheral blood total leukocyte response was estimated on
the basis of 100 cells counted in blood smears (Basten et al., 1979)
Ruitenberg
et
al.,
1977).
Cells were
stained with Wrights stain
(Aldrich Chemical Co.,Inc., Manual of Histologic and Special Staining
Techniques, 1949) and reported as
monocytes),
%
neutrophils, and
%
%
mononuclear cells (lymphocytes and
eosinophils.
36
PCA assay for worm-specific IgE
PCA tests were used to assay worm-specific IgE in the sera of H,.
diminuta infected mice.
Blood was obtained from the retro-orbital
plexus with a heparanized Pasteur pipette while mice were under ether
anesthesia (Sakai et al., 1981).
Collected blood was allowed to clot
overnight at 4° C, then centrifuged to further separate the serum from
the cells (Hindsbo et al., 1982).
Serum was stored separately for each
animal at -20° C then pooled on the day of testing in equal amounts of
0.1 ml of serum from each animal.
Test sera collected from 5 LM and 5
W/Wv mice on days 16,21, and 28 PI, from 5 WZWvBM mice on days 21 and
28 PI, and from 3 of each C57BL and BALBZc group on day 21 PI were
assayed.
All tests were done once with the exception of tests on sera
collected on days 21 and 28 PI from LM and WZWv mice, which were tested
twice with the same response both times.
PCA tests were performed on female Holtzman rats, 4-5 months old,
weighing 280-405 gm (Ovary et al., 1975).
pentobarbital
Rats were anesthetized with
and injected intradermally on their backs with 0.1 ml
aliquots of test sera.
Test sera were aliquoted in two-fold serial
dilutions starting with undiluted serum up to a dilution of 1:256. 48
hours after passive
sensitization,
rats were
challenged with. IV
injection of 1.0 mg of tapeworm antigen (TWA; see below) in 1.0 ml of
phosphate buffered saline
(PBS) with 0.5% Evans Blue
1975; Hindsbo et al., 1982).
(Ovary et al.,
Intradermal injection sites were checked
30 and 45 min later (Hindsbo et al., 1982) for blue stained areas
indicative of increased vascular permeability caused by the release of
mediators from sensitized mast
cells.
A circle of
dye
3-5 mm
in
I
37
(Hindsbo et al., 1982).
diameter is considered a positive reaction
Results from PCA tests are typically recorded as the reciprocal of the
maximum
serum dilution giving a positive reaction
1985).
A value of O was assigned to any test in which no reaction was
detected using undiluted serum.
were done using
brasiliensis
Positive
(Kamiya et
al.,
controls for PCA reactions
brasiliensjs antigen with sera collected from N.
infected
rat s , which
is known to give a strong PCA
reaction (Crowle and Reed, 1981).
Tapeworm
antigen
(TWA)
was
prepared
from adult H ie diminuta
recovered from the intestines of infected rats (Hindsbo et al., 1982;
Isaak,
1983).
Worms were soaked overnight in PBS, ground in a glass
tissue homogenizer for approximatly
I hr,
then gently
stirred on a
magnetic stirrer at 4° C overnight.
The homogenate was centrifuged at
4° C for 30 min at 11 ,500 rpm (2180Oxg), the supernatant removed and
lyophilized,
then
reconstituted
to
Img/ml
PBS for injection.
N.
brasiliensis antigen was prepared by collecting adult worms from the
intestines of infected rats.
Collected worms were ground with a glass
tissue homogenizer and centrifuged for 10 min at 1500 rpm (371xg).
N.
brasiliensis antigen was injected at a calculated dose of 2000 worm
equivilants per ml (Ha et al., 1986).
Statistical analysis
Standard deviations
(SD)
from the means and probabilities
values) of significance were determined
except
in the
when the sample size was >.3 »
case of the T l confusum data where
analyses were done on sample sizes of 2 (Table 6).
+/-
(p
signs on Tables and by error bars on Figures.
some statistical
SD are indicated by
Error bars which
38
extend beyond adjacent curves in figures occur in respective order to
data
points,
except where otherwise
(Figs. 5, 78, and 15).
indicated by
two separate bars
Results were first analyzed by a Student’s T
test (MSU Stat, version 2.2, Richard E. Lund, Montana State University,
Bozeman,
MT;
Snedecor and Cochran,
1976)
then further analyzed
for
significance by the Bonferonni T-test method, at p <_ .0125, .01 or .007
with a simultaneous significance of .05 for 4, 5 , or 7 statements.
The
Bonferonni T-test method takes into account simultaneous error rates
when estimating a number of mean responses (statements) corresponding
to vector X (days PI).
With this statistical analysis the confidence
intervals are lowered by claiming significance at p = .05 if p <_.05/k
(k = number of statements)
(Meter et al.,
1985;
Department
Montana
University,
of
Mathmatics,
personal communication).
State
Martin Hamilton,
Bozeman,
MT,
39
RESULTS
Littermate. W/Wv . and W/Wv bone marrow repaired mice
Intermediate host; Tenebrio politae
Worm recovery
Mice inoculated with 3 cysticercoids of
diminuta (intermediate
h o s t , Tj_ molitae) were killed at different intervals PI and their
intestines were checked for the presence of adult worms.
Some mice
from all groups, including the L M , were infection positive on day 6/7
PI (Table I).
By day 10 PI, all littermate mice were worm negative.
W/Wv and WZWvBM were 100% positive for infection on days 10 and .
12 PI, and on day 12 PI, respectively.
On day 12 PI, WZWv mice had a
93? REC, whereas 58? REC occurred in WZWvBM mice, and on day 16 PI WZWv
had a 30 ? REC where as a 13 ? REC occurred in WZWvBM mice.
On day 21
PI, both groups had a 13 ? REC of worms. These data indicate that worm
rejection occurs in a similar overall pattern from these two groups of
mice, but is slower at certain intervals, such as on days 12 and 16 PI,
from unrepaired WZWv mice.
The gradual loss of worms from WZWv mice
also indicates that the 3 worm burden is lost over time, rather than
simultaneously from each animal.
The median survival time, defined by Befus (1975) as the first
day on which >_ 50? of the worms administered to a group of mice have
been lost, was also used to compare the time of worm rejection.
survival
Median
time indicates that rejection of worms from both WZWv and
WZWvBM mice occurred on day 16 PI (Table I).
In contrast, control
40
Table I.
Percentage of mice positive for adult worms it POS) and
percentage 0f cysticercoids recovered as adult worms ($ REG)
at different intervals post-inoculation (PI) of littermate
(LM), ti/Hv , WZtiv bone marrow repaired (WZWvBM), and control
C57BL nude (C57BL NU) mice with 3 cysticercoids of
diminuta.
DAY PI
-GROUP
N
% POS
% REC
6/7 I
LM
W/Wv
W/WVBM
CS7BL NU
10
10
5
2
50
60
100
50
27
27
47
50
10
LM
Wwv
C53BL NU
5
5
2
0
100
100
0
100
50
12
iii
w/wv
W/WvBM
5
5
4
Q
100
100
0
93
58
16
LM
W/.WV
W/WvBM
C57BL NU
10
10
5
2
0
40
20
50
0
30
13
33
21
LM
W/Wv
W/WVBM
C57BL NU
10
10
5
2
0
30
20
0
0
13
13
0
24
LM
W/Wv
3
2
0 .
0 .
0
0
data from day 6/7 PI are considered minimal because small
size of worms and non-rigorous handling of tissue made
full recovery difficult.
41
C57BL nude mice rejected
diminuta earlier (median survival time of
10 days PI.
Worm growth
The mean length (cm) of H l diminuta worms recovered on days 6/7
PI was small
(3.5-11.0 cm)
in all groups of mice
compared to worm
lengths on subsequent days PI (Fig. I, Table 9; Appendix).
A striking
increase in the.length of worms occurred from day 10 PI onwards in both
W/Wv and WZWvBM mice,
reaching mean maximums of 93 cm and
respectively, on day 16 PI.
interval
in the control
100 cm,
Worms also grew in length during this time
C57BL nude mice,
but to only one-half the
length reached by worms recovered from the WZWv groups.
The mean dry weights (mg) of recovered worms appeared to increase
in correlation with worm length on days 12 and 16 PI for WZWv , WZWvBM
and control C57BL nude mice (Fig. 2, Table 9; Appendix).
On day 21 PI,
the mean
mice remained
dry
weight
of
worms
recovered
from WZWv
constant, while the mean length increased (Fig.
I).
Worms recovered
from the WZWv mice on day 21 PI appear shorter and heavier (Fig. 3)•
Mean
dry weights
considerably,
for worms
of
similar mean
length varied
particularly for the larger worms recovered on days 12
and 16 PI. Dividing the worms arbitrarily into small (1-10 cm recovered
on days 6/7 PI), medium (10-60 cm recovered on days 6/7 through day 12
PI) and large (60-138 cm recovered on day 12 through day 21 PI), dry
weights ranged from 0.5 to. 3-0 mg for small worms, 5-10 mg for medium
worms and 10 to 138 mg for large worms.
DAYS PO S T - I N O C U L A TIOS
I. Mean length (cm) of Hie dlmlnuta worms recovered from llttermate
( A ), W/Wv ( S )1 W/Wv bone marrow repaired ( O ), and control
C57BL nude ( O ) mice at different Intervals post-inoculation.
Values represented In this figure are listed In Table 9
(Appendix).1 2 3
1 . values represent means of combined lengths of all
worms/positive mouse in a group.
2. error bars indicate SD when the number of worm positive
mice in a group was >_ 3»
3 . symbols occur on the origin line of the x-axis when no
mice were worm positive.
43
DAYS P O S T - I N O C U L A T I O N
Fig. 2.
Mean weight (mg) of Hi diminuta worms recoved from littermate
(A), W/Wv ( S )1 W/Wv bone marrow repaired ( □ ) and control
C57BL nude ( O ) mice at different intervals post-inoculation.
Values represented in this figure are listed in Table 9
(Appendix).1 2 3
1. values represent means of combined weights of all
worms/positive mouse in a group.
2. error bars indicate SD when the number of worm positive
mice in a group was >. 3»
3 . symbols occur on the origin line of the x-axis when no mice
mice in a group were worm positive.
44
Fig. 3 .
Photograph of H2. dlmlnuta worms recovered from littermate
(LM), W/Mv , and control C57BL nude (BLN) mice on days 6, 10,
16, and 21 post-inoculation (PI). The arrow at LM, day 6 PI,
is pointing to a single small worm.
45
Hiatological observations
The distribution of MMC in four sections of littemate intestine
(Section
I, primarily duodeum,
Sections 2-4,
primarily jejunum)
was
examined for each day PI (Table 2).
Table 2.
MMC/10 VCU in four sections of the small intestine of
representative tL_ diminuta infected littemate mice
shows a small degree of regional differences.
Subsequent MMC counts and all intestinal eosinophil
counts for experiments were done in SECTIONS 2 or 3
to standardize counts in an area of greatest worm
attachment (Andreassen et al., 1978)^.
DAYS PI
SECTION I
(0-17)
duodenum
SECTION 2
(17-30%)
jejunum
SECTION 4
(43-56%)
jejunum
SECTION 3
(30-43%)
jejunum
7
5.6
12.0
12.0
9.4
12
19-0
21.6
17.3
ND2
16
ND
61.8
57.0
50.4
21
ND
111.0
79.6
81.0
I. five mice were sampled on days 7 and 16, three mice were
sampled on days 12 and 21.
2. ND = not done.
There was little variation in the number of mast cells in the
four sections,
higher
in most
although counts from Sections 2 and 3 were slightly
cases.
Taking this into consideration along with
reports that the attachment of H l diminuta
jejunal
intestine
subsequent
(Befus,
counts .of
Sections 2 or 3-
MMC
1975;
and
occurs primarily in the
Andreassen
et
al.,
1978),
all
intestinal eosinophils were done in
46
MMC accumulated in the small intestines of LM and WZWvBM mice,
but not in the small intestines of WZWv or CS7BL nude mice (in this
experiment) in response to Hjl diminuta infections
Appendix).
mice
(Fig. 4, Table 9;
MMC increased significantly (p = .0000) to a. peak in LM
on day 21 Pl compared to day 0 values,
but did not increase
significantly at any other sample time PI (significant at p £ .007 with
a simultaneous significance of .OS for 7 statements, days 0 , 6Z7, 10,
12, 16, 21, and 24 PI). Although not significantly higher than day 0,
an early peak in MMC numbers appears to occur on day 10 PI in LM mice.
The greatest mean number of MMC was observed in both LM and WZWvBM mice
on day 21 PI, followed by a substantial drop in LM mice on day 24 PI.
The
accumulation
of
MMC
in LM mice
was
not
significantly
different than the accumulation in WZWvBM mice on days 6Z7, 12, and 16
PI (p = .2245, .4889,
and .5805 comparing LM to WZWvBM on days 6Z7,
12, and 16, respectively; p = .0464 on day 21 PI; significant at p <L
.0125 with
a simultaneous significance of
.05,
4 statements).
The
slight increase in MMC in the unrepaired WZWv mice on day 21 PI was not
significant (p = .1092 ) compared to day 0 for these mice.
A few MMC
(0-5Z1Q VCU) were observed in the intestines of C57BL nude mice (Table
9; Appendix)‘
Eosinophils accumulated in the intestines of all four groups of
^ mice
in
response
to H j, diminuta
(Fig.5,
Table
9;
Appendix).
A
significant increase in intestinal eosinophils occured in LM mice on
day 10 PI compared to day 0 (p = .0000; significance; p <_ ,007 with a
simultaneous
signifcance
of
.05 for 7 statements), followed by
substantial decline on day 12 PI.
A later peak of intestinal
a
M M C /10 VCU
47
DAYS
Fig. 4.
POST-INOCULATION
Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of littermate ( ▲), W/Wv (•) ,
and W/Wv bone marrow repaired ( O ) mice at different intervals
post-inoculation with 3 cysticercoids of
diminuta. Error
bars indicate SD. Values represented in this figure and values
for control C57BL mude mice are listed in Table 9 (Appendix).
48
DAYS P O S T - I N O C U L A T I O N
Fig. 5.
Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of litteroate ( A ), VIZWv (•), WZViv
bone marrow repaired ( D ) 1 and control C57BL nude ( O ) mice at
different intervals post-inoculation with 3 cysticercoids of
H. dlmlnuta. Error bars indicate SD. Values represented in
this figure are listed in Table 9 (Appendix).
49
eosinophils in LM mice on day 21 PI in LM mice was also significant
compared to day 0 (p = .0024)
and coincided with the greatest mean
number of eosinophils observed in the other three groups.
The number of eosinophils observed for LM and.WZWvBM mice was not
significantly different at any interval of time PI ( p = . 7 3 4 2 ,
. 4 3 1 5 , and
21
PI,
.3934,
.1 7 7 0 comparing LM to WZWvBM mice on days 6 Z 7 , 1 2 , 1 6 , and
respectively;
significant at p <_ . 0 1 2 5
with
a simultaneous
significance of . 0 5 , 4 statements). The number of eosinophils observed
for WZWv mice was significantly lower than the number observed in LM
mice on days 6 Z 7 and 10 PI
(p = . 0 0 1 0 and . 0 0 0 0 comparing LM to WZWv
mice on days 6 Z 7 and 10 PI, respectively; significant at p <_ . 0 0 7 with
a simulataneous significance of . 0 5 , 7 statements; days 0 , 6 Z 7 , 1 0 , 1 2 ,
16,
2 1 , and 2 4 PI).
Blood cell response
Eosinophils
in the peripheral blood
(EOSZmm^) dropped in L M ,
WZWv , and WZWvBM mice on day I PI, then rose in all three groups on day
3 PI (Fig. 6 , Table 10; Appendix),.
Levels dropped and fluctuated on
days 5 through 9 BI, then began to rise again on day 11 PI in LM and
WZWvBM mice and on day 13 BI in WZWv mice to reach peak numbers in all
three groups on days 13 through 17 BI. These increases in peripheral
blood eosinophils occurred just prior to the occurrence of peak numbers
of eosinophils in the intestines on day 21 BI (Fig. 5)'
Beripheral blood eosinophils were sampled in C57BL nude mice on
days 5,9,15, and 21 BI.
On days 5 and 21 BI, blood eosinophil levels
in control 057BL nude mice were similar to those of LM and WZWvBM mice.
50
w
700
DAYS
Fig. 6.
POST-INOCULATION
Mean number of eosinophils (EOS) per cubic millimeter (mm3) 0f
peripheral blood collected from littermate ( A ), V/Mv ( • ),
and W/Wv bone marrow repaired ( O ) mice on alternate days
post inoculation with 3 cysticercoids of Hjl diminuta. Blood
was collected from C57BL nude mice on days 5, 9» 15, and 21
PI. Values represented in this figure are listed, with SD, in
Table 10 (Appendix).
51
On day 9 PI the level of blood eosinophils dropped to 100 EOS/mm^ an(^
on day 15 BI it rose to 950 EOS/min^ in the C57BL nude mice.
level of eosinophils in the blood of C57BL nude
The high
mice on day 15 PI
occurred prior to the occurrence of highest numbers of eosinophils in
the intestines on day 21 PI.
Counts of eosinophils in the peripheral blood are reported to be
highly variable in both infected and non-infected animals (Oglivie et
al., 1978).
Even though blood samples were obtained from animals at a
designated time each sample day to minimize
1974),
considerable
variation
in
the variation
blood, eosinophil
occurred, even in a single littermate mouse (Fig. 7).
(Colley,
counts
still
Such variation
in eosinophil counts was typical of all sampled animals, regardless of
strain.
Differential
leukocyte
counts
revealed
an
increase
in the
percentage of eosinophils in WZWvBM mice on days 15 and 17 PI with a
concomitant decrease in the percentage of mononuclear cells (Table 3)*
There is no obvious pattern to most of the fluctuation in the relative
numbers of mononuclear cells, neutrophils and eosinophils in the blood
of Hjl diminuta infected LM, WZWv , and WZWvBM mice.
Worm specific IgE
PCA
tests
for
worm-specific
IgE in the sera of
infected mice were negative for all days tested
(Table
diminuta
4).
Rats
injected with the sera of W l brasiliensis infected rats and challenged
with N l brasiliensis antigen gave positive PCA tests, confirming the
reliability of the test.
52
M
500
DAYS P O S T - I N O C U L A T I O N
Fig. 7.
Eosinophils (EOS) per cubic millimeter (mm3) 0f
peripheral blood collected from a single representative
littermate mouse on alternate days post-inoculation with 3
cysticercoids of
diminuta.
-Table 3.
Differential leukocyte counts of 100 cells in Wright’s stained blood smears made from blood _
collected from LM, ,W/W,V» and w/WVBM mice on alternate days post-inocuation and starting on
day -I. Leukocyte counts are reported as % mononuclear cells, % neutrophils and % eosinophils.
LEUKOCYTE'
GROUP
DAY P O S T - I N O C U L A T I O N
-I
% MONONUCLEAR
CELLS
% NEUTROPHILS
7o
EOSINOPHILS
I
3
5.
7
9
11
13
15
17
19
21
LM
87.8
91.2
86.0
84.2
81.5
81.8
82.6
80.0
84.2
86.4
83.0
79.6
W/WV
88.2
87.4
82.2
85.2
89.3
84.8
84.8
81.2
82.2
88.4
82.8
73.0
W/WVBM
90.7
91.0
87.0
87.2
87.4
85.3
81.6
82.6
77.8
75.8
75.2
80.0 '
LM
10.0
7.6 ‘10.0
10.4
14.0
11.5
10.2
10.6
8.0
8.4
10.2
13.2
W/Wv
10.8 /10.2
16.0
13.0
8.0
13.0
14.4
13.6
13.0
7.0
11.6
23.0
8.0 •11.0
10.8
13.2
15.2
11.8.
11.0
10.4 . 16.2
12.2
W/WvBM
5.3
7.0
LM
2.6
.1.6.
4.2
5.8
3.6
7.0
7.2
9.4
7.0
5.2
6.8
7.2
WzWv
1.0
2.4
1.8
1.6
3.0
1.8
0.8
5.5
5.0
•4.6
5.6
4.0
W-/WvBM
4.0
2.0
4.7
3.2
3.8
. 3.0
3.8 . 5.6
12.0
13.8
8.6
7.0
54
Table 4.
Passive cutaneous anaphylaxis tests for the presence of wormspecific IgE in the sera of littermate (LM), WZWVf and WVWv
bone marrow repaired (WVWvBM) mice collected on days 16, 21,
and 28 post-inoculation (PI) with 3 cysticercoids .of
Hi diminuta. Results are reported as the highest dilution
giving a positive reaction with a value of 0 indicating a
negative reaction. Antigen challenge was given 48 hours after
serum injection. The sera from
brasiliensis infected rats
was used as a positive control for the test.
SERUM TESTED
ANIMAL
DAfPI
SERUM DILUTIONS
TIME POST-CHALLENGE
30 MIN
45 MIN
1:256
1:0
. 1:0 - 1:256
0
0
0
0
LM
W/Wv
W/WvBM
1:0 — 1:256
1:0 - 1:256
1:0 - 1:256
0
0
0
C
0 .
0
LM
■ W/WV
W/WvBM
1:0 — 1:512
1:0 - 1:512
1:0 - 1:256
0
0
0 -
0
0
o
1:0 — 1:512
1:512
1:0
64
32
16
LM
W/Wv
21
28
Control Rat I
Control Rat 2
21
.
Littermate and WVWv mice
Intermediate host: Tribollun confuaup
Worm recovery
Since the intermediate host
source could affect the definitive
host response, data derived from the use Ti molitae and Ti confusum as
sources of Hi diminuta cysticercoids Were compared.
LM and WVWv mice
inoculated with 3 cysticercoids of Hi diminuta were killed at different
intervals PI and their intestines were
adult worms.
checked for the. presence
of
55
The lack of worm recovery on day 6 PI for both groups of mice
(Table 5) probably reflects the small worm
size
and
the
recovery
technique used rather than an actual total absence of worms. On days
12, 18, and 24 PI, all LM mice remained worm negative, indicating that
LM mice definitely rejected Hi diminuta by day 12 PI, while W/Wv mice
remained worm positive until day 18 PI.
Table 5.
The percentage of mice positive for adult worms {% POS) and
the percentage of injected cysticercoids recovered as adult
worms (% EEC) in littermate (LM) and W/Wv mice with the
intermediate host source Tribolium confusum.
DAY PI
GROUP
%
POS
%
EEC
61
LM
W/Wv
0
0
0
0
12
LM
. W/WV
.0
80
0
47
18
LM
W/WV
0
40
0
13
24
LM
WZWv
0
0
0
0
The median survival time indicated that W/Wv mice inoculated with
H. diminuta derived from the intermediate host T . confusum rejected the
parasite on day I2 PI (Table 5).
Worm growth .
Worms recovered from W/Wv mice reached a maximum mean length of
88.7 cm on day. 12 PI and then decreased in mean length on day 16 PI to
63.3. cm (Fig. 8, Table 11; Appendix), probably due to destrobilation
(Hopkins et al., 1972).
Mean worm weights increased over time as the
worm length decreased (Fig. 8).
56
z u
U Z
DAYS POS I N O C U L A T ION
Fig. 8.
Mean length (cm) and weight (mg) of IL diminuta worms
recovered from litteraate (A) and W/Wv ( • ) mice at different
intervals post-inoculation with 3 cysticercoids derived from
the intermediate host
confusum. Values represented in this
figure are listed in Table 11 (Appendix).1 2 ^
1. Values represent means of combined lengths of all
worms/positive mouse in a group.
2. Error bars indicate the SD when the number of worm positive
mice was > 3»
3. Symbols occur on the origin line of the x-axis when 0% of
the mice in a group were worm positive.
57
Histological observations
Mucosal mast cells (MMC) accumulated in highest mean numbers in
LM mice on day 24 PI (Fig. 9, Table 11; Appendix).
in
MMC
accumulation
in LM mice
on day
An earlier "peak"
12 PI may not have been
significantly higher than the levels of MMC which accumulated on days 6
and 18 PI.
The level of MMC present in the intestines of W/Wv mice
remained low during the course of the experiment.
The
mean
number
of
intestinal
eosinophils
in LM mice
Was
consistantly higher than the mean number of intestinal eosinophils in
W/Wv mice
(Fig.
10, Table 11; Appendix).
increased in both groups
on
intestinal eosinophils in LM
day
21
PI.
The number of eosinophils
An earlier
increase in
mice on day 12 PI is only not!cable
in
terms of the high standard deviation from the mean.
Tenebrio colitae vs. Triboliun confusun, Intermediate hosts
Mean lengths of worms recovered from W/Wv mice were very similar at
early and late intervals PI with the two different intermediate hosts,
however,
the time course of growth was different. Worms from the T.
molitae source (1st experiment) reached a maximum length (in the W/Wv )
on day 16 PI
(Fig.
I) while
experiment) reached a maximum
worms from the T^ confusum source (2nd
length on day
12
PI
(Fig. 8).
Mean weights of worms recovered from W/Wv mice increased with
both intermediate host sources with increasing time PI up to day 16,
but decreased with Ti molitae.on day 21 PI (Fig. 2) while continuing to
increase with Ti confusum on day 18 PI (Fig. 8). For both intermediate
hosts, littermate mice rejected the worm earlier, by day 10 PI with Ti
58
•—1 60
DAYS POST-INOCULATION
Fig. 9.
Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of littermate ( ▲) and w/w ( • )
mice at different intervals post inoculation with 3
cysticercoids of EU dlminuta derived from the intermediate
host T\ confusum. Error bars indicate SD. Values represented
on this figure are listed in Table 11 (Appendix).
59
200
180
160
140
I 120
c
^ 100
Cfi
C
M
80
60
40
2C
O
4
8
12
16
20
24
DAYS POST-INOCULATION
Fig. 10.
Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of littennate ( A ) and W/Wv ( • ) mice
at different intervals post inoculation with 3 cysticercoids
of H l diminuta derived from the intermediate host T l
copfusum. Error bars indicate SD. Values represented on this
figure are listed in Table 11 (Appendix).
60
molitae
and
by day
12 PI with T l confusum. than W/Wv mice, .which
rejected the worm by day 24 PI in both experiments.
The MMC and intestinal eosinophil
responses wer e .significantly
different at a few times PI for both LM and W/Wv mice with the two
intermediate host, sources
(Table 6).
reflect, perhaps for the most
part,
These
significant differences
the different time courses of
increases in MMC and eosinophils with T l molitae or T l confusum.
example,
the maximum numbers of MMC occurred in the intestines
For
of
littermate mice in the case of T l molitae on day 21 PI and in the case
of T l confusum on day 24 PI (at levels of 94.0 MMC/10 VCU and 96.4
MMC/10 VCU, respectively, Figs. 4 and 9).
Table 6.
.
Probabilities (p values) of the significant difference
between the MMC and intestinal eosinophil (EOS)
responses of littermate (LM) and W/Wv mice to
H.diminuta derived from two different intermediate host
sources; Tenebrio molitae and Tribolium confusum.
Probabilities were determined when sample size was at
least three animals in both experiments.
DAY PI
T . molitae
T. confusum
ANIMAL GROUP
MMC
EOS
p values
6
LM
WZWv
12
12
LM
WZWv
16
18
LM
WZWv
.9864
.5861
24
24
LM
WZWv
.OOOO1 .0276
.71562 .OOOO2
6/7
.0154
ND
.OOOO1
.00311
. .0169 •3506
.00381 .3113
.0323
.4671
.
1. significant at p <_ .0125 with a simultaneous significance of
.05 for statements, days 6/7-6, 12, 16-18, and 24 PI.
2. sample size was two in T l molitae experiment.
61
Overall, the cellular response with Tenebrlo molitae was earlier
and
lower
than
the
cellular response with Tribolium confusum,
maximum growth of the
diminuta worms was delayed.
and
The patterns of
MMC and intestinal eosinophil accumulation in LM and W/Wv mice were, in
many
ways,
similiar
in the
two
experiments.
MMC showed biphasic
increases in LM mice with both Tenebrio molitae and Tribolium confusum.
MMC occurred in W/Wv mice to a similiar degree with both Ti molitae and
Ti
confusum.
Intestinal, eosinophils showed a pronounced biphasic
increase in LM mice with Tenebrio
molitae. on days 10 and 21 PI, while
a late increase intestinal eosinophils, on day 24 PI, occurred in LM
mice with T. confusum.
Nude mice
Worm recovery
Two
stains of athymic nude mice,
littermates
were
compared
for
inoculations with Hi diminuta
their
C57BL and BALB/c,
response
to
and their
3 cysticercoid
(intermediate host; Ti molitae). Both
C57BL and BALB/c littermate mice rejected Hi diminuta by day 12 PI, as
expected for normal littermates (Table 7) •
rejection of the parasite from
Based on
%
POS data, the
the C57BL nude appeared to be slightly
slower than rejection from the BALB/c nude, i.e. the worm was present
in one C57BL nude mouse on day 21 PI, but was absent from the BALB/c
(Fig.
14).
Based on median
survival time, both .057BL
nudes rejected the parasite by day 16 PI, with a 33%
both strains (Table 7)•
and BALB/c
recovery from
62
Table 7.
Percentage of mice positive for adult worms (% POS) and
percentage of cysticercoids recovered as adult worms (% RKC)
at different intervals post-inoculation (PI) of C57BL
littermate (LM), C57BL nude (NU), BALB/c littermate (LM), and
BALB/c nude (NU) mice. Three mice from each group were killed
on each sample day.
DAY PI
71
GROUP
C57BL LM
C57BL NU
BALB/c LM
BALB/c NU.
% POS
100 .
■ 66 .
33
0
% REC
33
11
33
0
12
C57BL LM
C57BL NU
BALB/c LM
BALB/c NU
0
100
0
100
0
■ 56
0
78
16
C57BL LM
C57BL NU
BALB/c LM
BALB/c NU
0
67
0
33
0
33
0
33
.21
C57BL LM
C57BL NU
BALB/c LM
BALB/c. NU
0
33
0
0
0
.11
0
0
I. data from day 7 PI are considered minimal because small
size of worms and non-rigorous handling of tissue made
full recovery difficult.
63
Worm growth
The mean length (cm) of recovered worms on day 7 PI ranged from
2.4 cm for C57BL littermates to 13.4 cm for BALB/c littermates (Fig.
11, Tables 12 and 13; Appendix).
The average worm length for C57BL
nudes on day 7 PI was 5.7 cm, no worms were recovered from BALB/c nudes
on day 7 PI, mice in this group were presumably infection positive on
this day with undeteetably small worms.
An increase in worm length in the nudes on days 12 and 16 PI was
particularly dramatic in the BALB/c
Appendix).
(Fig.
11,
Tables
12 and
13,
Three worms recovered from one positive BALB/c nude on day
16 PI had a (combined total) length of 111.2 cm.
Two C57BL nudes were
positive on day 16 PI for two worms each which averaged in (combined
total) length to only 37.1 cm.
The pattern of
diminuta rejection
from the C57BL and BALB/c nudes appears to be very similiar, but the
growth of the worm appears to be greater in the BALB/c.
The mean weights (mg) of worms recovered from C57BL and BALB/c
nudes (Fig. 12, Tables 12 and 13; Appendix) was considerably lower than
the mean weights of worms of comparable lengths recovered from WZWv
mice.
host
A 100 cm total worm recovered from a W/Wv mouse, intermediate
T l molitae,
weighed 56.0 mg. The same length worm from a W/Wv
mouse, intermediate host T l confusum, would have a calculated weight of
38.1 mg. The 111.2 cm total worm recovered from the BALB/c nude weighed
7.0 mg (the same length worm, 100cm, would have a calculated weight of
6.3 cm).
Also in constrast to the W/Wv mice, the mean weights of worms
recovered from C57BL and BALB/c nudes decreased over time (Fig. 12)
64
DAYS POST-INOCULATION
Fig. 11.
Mean length (cm) of
dlminuta worms recovered from
C57BL littermate (A), C57BL nude (•), BALB/c littermate
( □ ), and BALB/c nude ( O ) mice at different intervals postinoculation. Values represented in this figure are listeo in
Tables 12 and 13 (Appendix).1 2 3
1. values represent means of combined lengths of all
worms/positive mouse in a group.
2. error bars indicate SD when the number of worm positive
mice was >_ 3«
3 . symbols occur on the origin line of the x-axis when 0%
the mice in a group were worm positive.
65
DAYS POST-INOCULATION
Fig. 12.
Mean weight (mg) of
dlmlnuta worms recovered from
C57BL littermate (A), C57BL nude, (•), BALB/c llttermate
(□), and BALB/c nude ( O ) mice at different intervals postinoculation. Values represented in this figure are listed in
Tables 12 and 13 (Appendix).1 2 3
1. values represent means of combined weights of all
worms/ positive mouse in a group.
2. error bars indicate SD when the number of worm positive
mice was >_ 3 •
3. symbols occur on the origin line of the x-axis when 0 %
of the mice in a group were worm positive.
66
rather than remaining constant
(T. molitae, WZWv mice) or increasing
(T. confusum, WZWv mice) (Figs. 2 and 8).
Histological observations
MMC were
observed in the
intestines of both
C57BL arid BALBZc
littermates, and in surprisingly high numbers in the intestines of both
C57BL and BALBZc nudes (Figs. 13 and 14, Tables 12 and 13; Appendix).
The number of MMC observed in C57BL and BALBZc nude mice was considered
high since nude mice are commonly described as completely lacking or
having only a few MMC
(Ruitenberg and Elgersma, 1976; Andreassen et
al., 1978; Crowle and Reed, 1984).
75.0 MMCZ
The maximum mean number of MMC was
10 VCU on day 21 PI for C57BL nudes
MMC Z 10 VCU
on day
16 PI for BALBZc nudes
reached peak numbers
of
150.7
MMCZ10VCU
(Fig.
(Fig.
and
14).
164.0
13) and
58.0
Littermates
■ .
MMCZ 10 VCU,
respectively.
Since the lack of MMC in nude mice is related to the absence of a
functional
examined
tissue.
thymus,
the tissue in the mediastinum of nude mice
histological
for the possible presence
of
normal
was
thymus
None was found.
The MMC in the intestines of neither the C57BL nor the BALBZc
nudes increased significantly during the course of the experiment, in
spite of the apparent increase of MMC on day 21 PI in the C57BL nude
■
(C57BL nude: p = .3815, 1.0, .1963, and .1910 for days 7, 12, 16, and
21
PI,
respectively,
compared to day 0; BALBZc: p =
.0633,
.1516,
.0419, and .5922 for days 7, 12, 16, and 21 PI, respectively, compared
to day 0; significant at <_ .01 with a simultaneous significance of .05,
■
5 statements, both groups).
In contrast, the increased numbers of MMC
I
MMC/IO VCU
67
Fig. 13. Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of C57BL littermate ( A ) and
nude ( • ) mice at different intervals post-inoculation with 3
cysticercoids of H l diminuta. Error bars indicate SD. Values
represented in this figure are listed in Table 12 (Appencix).
68
DAYS POST-INOCULATION
Fig. 14.
Mean number of mucosal mast cells (MMC) per 10 villus crypt
units (VCU) in the intestines of BALB/c littermate ( D ) and
nude ( O ) mice at different intervals post-inoculation with 3
cysticercoids of
diminuta. Error bars indicate SD. Values
represented in this figure are listed in Table 13 (Appendix).
69
in C57BL littermates were significant on day 21 PI (p = .0032 compared
to day 0) and in the BALB/c littermates were significant on day 16 PI
(p = .0028 compared to day 0).
nude
intestines
was
Since the number of MMC observed in the
unusually
possibility that the
h i g h , and
in consideration of the
diminuta parasite was intitating the response
in a thymus independent manner, groups of CS7BL and BALB/c nude mice
were infected with either Hi diminuta or Ni brasiliensis.
Nude mice
have not developed a MMC response to the nematode Ni brasiliensis in
the past (Crowle and Reed, 1981), therefore, if Hi diminuta was unique
in eliciting a thymus-independent MMC repsonse, the response should not
be
evident
in the
nude mice infected with N. brasiliensis.
Upon
histological examination, MMC were observed in the intestines of both
Hi diminuta and Ni brasiliensis infected CS7BL and BALB/c nude mice
(actual counts not done).
The mean number of eosinophils observed in the intestines of
C57BL littermate mice increased significantly on day 12 PI (p = .0000,
compared to day 0), but- otherwise remained at levels similar to the
levels of intestinal eosinophils observed in C57BL nude mice, which did
not increase significantly at any time interval PI (Fig. 15, Table 12;
Appendix).
The mean number of eosinophils increased in the intestines
of both BALB/c littermates and nudes in a biphasic pattern (Fig. 16,
Table
13; Appendix),
peaking in the littermates on
significant level (p =
day
7 PI to
a
.0090 compared to day 0; significant at <_ .01
with a simultaneous significance, of .05, 5 statements; days 0, 7., 12,
16, and 21 PI).
Eosinophil numbers dropped significantly below day 0
numbers in both BALB/c littermates and nudes on day 21 PI (BALB/c L M ; p
70
DAYS POST-INOCULATION
Fig. 15.
Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of C57BL littermate ( A ) and nude
( • ) mice at different intervals post-inoculation with 3
cysticercoids of
diminuta. Error bars indicate SB. Values
representec in this figure are listed in Table 12 (Appencix).
EOS/ 10
71
DAYS POST-INOCULATION
Fig. 16.
Mean number of eosinophils (EOS) per 10 villus crypt units
(VCU) in the intestines of BALB/c littermate ( □ ) and nude
( O ) mice at different intervals post-inoculation with 3
cysticercoids of Hi dimlnuta. Error bars indicate SD. Values
represented in this figure are listed in Table 13 (Appendix).
72
=
.0000,
BkLB/Q
NO;
p =
.0061)
Significant
peaks
of
intestinal
eosinophil accumulation occurred in both strains of littermates but was
lacking in both strains of nudes, and the pattern of eosinophil numbers
was different for each strain.
Blood cell response
Peripheral blood eosinophils also varied between the CS7BL and
BALB/c mice (Figs. 17 and 18, Table 14; Appendix).
Peripheral blood
eosinophil numbers peaked on day 10 for C57BL littermates and on day 16
for C57BL nudes, otherwise the pattern of eosinophilia in the C57BL
mice was similiar (Fig.
17).
The peripheral blood eosinophil level
increased in BALB/c littermates between days 10-14 PI, but was nearly
the same on early and late days PI as blood eosinophil levels in BALB/c
nudes, which did not appear to increase at any PI sample time (Fig.
18 ).
Worm specific IgE
Sera, collected from CS7BL and BALB/c littermate and nude mice on
day 21 post-inoculation were tested for Hi diminuta-specific IgE by PCA
tests in rats.
All sera were negative for worm-specific IgE (Table 8).
EOS/MM
73
DAYS POST-INOCULATION
Fig. 17.
Mean number of eosinophils (EOS) per cubic millimeter (mm3)
of peripheral blood collected from C57BL littermate ( A ) and
nude ( • ) mice on alternate days post-inoculation with 3
cysticercoids of Hi. diminuta, starting with day 0 (preinoculation). Values represented in this figure are listed
with SD in Table 14 (Appendix).
74
1300
1200
EOS/MM
1000
DAYS POST-INOCULATION
Fig. 18.
Mean number of eosinophils (EOS) per cubic millimeter (mm3)
of peripheral blood collected from BALB/c littermate (D) and
nude ( O ) mice on alternate days post-inoculation with 3
cysticercoids of H i. diminuta. starting with day 0 (preinoculation). Values represented in this figure are listed
with SD in Table 14 (Appendix).
75 ■
Table 8. PCA tests for the presence of worm-specific IgE in the
serum of C57BL littermate (LM), CS7BL nude (NU), BALB/c
littermate (LM), and BALB/c nude (NU) mice on day 21
post-inoculation (PI) with H2. diminuta. A value of 0
indicates a negative reaction.
SERUM TESTED
DAY PI
ANIMAL
SERUM DILUTIONS
TIME POST-CHALLENGE
45 MIN.
21
CS7BL LM
1:0 - 1:256
21
C57BL NU
1:0 - 1:256
0
21
BALB/c LM
1:0 - 1:256
0
21
BALB/c NU
1:0 - 1:256
0
.
0
76
DISCUSSION
Early increases in the numbers of MMC in the intestines of LM
mice on days 10 and 12 PI,
(Ti molitae. and Ti confusum experiments,
respectively), and of WZWvBM mice on day 21 PI, occurred in apparent
association with worm rejection indicating an involvement of the MMC in
the rejection mechanism.
The nature of MMC involvement in parasite
rejection may be varied, with the general effect of MMC mediators being
to render the intestinal microenvironment inhospitable to the parasite
(Kelly and Dineen, 1976; Harari et al., 1987).
A
limited
number
of
MMC
occurred
in
the
intestines
of
unrepaired WZWv mice, but did not increase significantly at any time PI
and were always fewer than MMC observed in LM and WZWvBM.
However, the
development and rejection of Hi diminuta was nearly identical in WZWv
and WZWvBM mice.
In both groups, worms grew to substantial sizes,
maximum growth occurred at the same time (day 16 PI), and the parasite
was retained in both up to day 21 PI.
It is evident that the presence
of MMC in increased numbers in the intestines of Hi diminuta infected
mice does not necessarily result in an accelerated rejection of the
parasite.
Later peaks of MMC accumulation in LM mice* on days 21 and 24 PI
(Ti molitae and Ti confusum experiments, respectively), occurred well
after parasite rejection from these animals and thus lend validity to
suggestions of alternative roles for MMC
parasites.
Some mast
in
the
host
response
cell-released mediators (histamine,
to
serotonin,
and proteases) may function to regulate the activity of other cells of
77
the host protective system, such as eosinophils and T lymphocytes, or
may function in the repair of parasite damaged tissue
(Ferguson and
Miller, 1979; Wasserman, 1979; Woodbury and Miller, 1982).
It must be noted that the observable increase in the number of
MMC usually occurred after the parasite was rejected, by day 10 PI from
LM
mice
and
after
day
21
PI
from
W/Wv
mice.
The
observable
accumulation of MMC has often been reported as occurring subsequent to
parasite rejection
(Wells,
1962;
Oglivie and L ove, 1974;
Miller, 1979; Alizideh and Murrel, 1984).
Nawa
and
Although this has led some
investigators to discount an involvement of MMC in rejection (Keller,
1971),
other investigators list
the possibility that a functionally
active MMC would, by nature, be degranulated, and therefore unstainable
at the time it is most active (Jones and Oglivie, 1971; Askenase, 1980;
Alzideh and Murrel, 1984).
The MMC response could be most observable when the stimulus for
degranulation, i.e. the parasite, was absent but while the stimuli for
development
of
lymphocytes,
the
were
cell,
still
i.e.
factors
present
in
released from activated T
the
intestinal
mucosa.
Immunospecific- or radio- labelling of different populations of mouse
intestinal cells (or cell products) at different time intervals post­
inoculation of a specific parasite antigen might provide a method of
testing the cellular interactions occurring with
the observable MMC
response.
Intestinal eosinophils accumulated in all groups of LM and W/Wv
mice.
In littermate mice, increases in intestinal eosinophils occurred
both early, especially with the Tenebrio molitae intermediate host, and
78
late.
The early increases in littermate mice occurred in temporal
association
with
parasite
rejection.
In W/Wv mice,
increases in
intestinal eosinophils occurred late in all cases and were coincident
with the late rejection of the parasite.
These temporal associations
indicate that eosinophils, as MMC, are involved in parasite rejection.
Eosinophils have been shown to adhere,
in vitro,
in the presence of
specific anti-sera, to the surface of Ni. brasiliensis adults and
mansoni
larvae.
In the
case
of
Si mansoni larvae;
eosinophils are cytotoxic to the parasite
(Oglivie
et
S.
the adherent
al . , 1977;
Butterworth et al., 1975).
The
coincident
accumulations
of
eosinophils and MMC in the
intestines of LM and WZWvBM mice indicate an interaction between these
two cell
types.
Many
studies
have
related
the
MMC
response
to
increased levels of eosinophils in parasite infected animals (Wells,
1962; Kelly and Oglivie, 1972; Kay, 1979; Dessein et al., 1981; Kamiya,
1985) .
This
eosinophil
interaction may be facilitated by the release
chemotaxic
factor of anaphylaxis
(ECF-A)
of an
from MMC which
attracts eosinophils to the site of a parasite infection (Kay, 1979) •
Recruited eosinophils may then function in parasite rejection or
regulate
MMC
through
the
deactivation
of
to
MMC-released mediators.
Eosinophil accumulation is not dependent upon the release of ECF-A from
mucosal mast
cells,
since eosinophils occurred in the intestines of
both WZWv and control C57BL nude mice which lacked significant numbers
of MMC.
Comparison of the dynamics of Hi diminuta infections in WZWv and
nude mice showed that worms generally grew to be longer and heavier in
79
the W/Wv (both repaired and unrepaired) and were retained for a longer
period of time.
The nature of the defect in both animals, W/Wv and
nudes, likely influences the effectiveness of the host response to the
tapeworm.
It
is
interesting that
the thymus deficient nude mice
appeared to be more refractive to the worm than the W/Wv . This seems to
indicate a more serious defect in the W/Wv , but the existence of a
strong immune potential has been clearly established in these animals
(Ha et al., 1986).
The stem cell-related defect in W/Wv mice may involve the absence
of factors which affect the function of the host immune system within
the W/Wv tissues.
the same level
Suda et al. (1985) demonstrated that W/Wv mice have
of mast
cell precursors in bone marrow,
blood,
and
spleen as.do normal littermates, and suggested that the W/Wv defect may
involve
the inability of precursor
cells,
such
precursor, to enter or differentiate in the tissue.
be
tested
by
isolating
and
labeling
cell
as
the mast
cell
This concept could
populations
from
the
intestines of normal littermates, providing these cells to the WVWv ,
then observing the intestinal response in WVWv mice exposed to specific
stimuli,
such
as Ni brasiliensis. T.
spiralis, and
Hi
diminuta
antigens.
Variations in the response of LM and WVWv mice to Hi diminuta
derived from two different species of intermediate host, T l molitae and
Ti confusum, indicate that the source of the infective larvae may be
significant in comparative studies of the definitive host.
Hi diminuta
worms derived from the intermediate host Ti confusum grew to a similar
size as worms derived from Ti molitae, but reached a maximup length
80
sooner and elicited stronger and later cellular responses.
Since the
differences observed with the two intermediate hosts may reflect strain
differences in the
diminuta source of infective eggs as well as, or
instead of, differences in the intermediate host, the use of a single
controlled
source of infective eggs could provide
more
conclusive
results.
Comparisons of the response to Hi diminuta infection in CS7BL and
BALB/c nude mice revealed similarities in the pattern of worm rejection
and development of MMC in these mice, while the lengths of recovered
worms differed.
The worms recovered from
twice
as
as
long
those
recovered
BALB/c nude mice were nearly
from
CS7BL nude mice.
This is
consistent with previous reports of Hi diminuta worms reaching lengths
of up to 70 cm in BALB/c nude mice (Isaak et al., 1975).
mice
with
Since nude
different genetic backgrounds have been shown to exhibit
differences in immune
system activities
(Nomura,
1984),
comparing
responses of the CS7BL nude and BALB/c nude to different parasites,
such
as
Hi
diminuta. may
be
useful
in evaluating the degree of
variation between different strains of nudes on the effect of a missing
thymus.
The most striking observation in the nude mice in this study was
the occurance of unusually high (for nudes) levels of intestinal MMC.
MMC reached levels of 75.0 MMC/10 VCU in the intestines of CS7BL nudes
and 58.0 MMC/10 VCU in the intestines of BALB/c nudes.
Although the
numbers of MMC did not increase significantly in either group at any of
the sample times PI (compared to day 0), the numbers of MMC observed in
the
i n testines
of
nude
mice
were
unexpectedly
high.
Other
81
investigators have reported MMC in. the intestines of nude mice, but at
much lower levels, e.g..2 MMC/10 VCU in non-infected BALB/c and C57BL
nudes
(Mayrhofer
and
Bazin,
1981)
and
17.7
MMC/
10
VCU
in
N.
brasiliensjs infected Swiss strain nudes (Levy and Frondoza, 1983).
MMC observed in the intestines of CS7BL and BALB/c nudes occurred
at
lower
levels
littermates*
than
those
observed
in
C57BL,
BALB/c,
and WZWv
but occurred at the same or higher levels than those
observed in WZWvBM mice (Figs. 4, 9, 13, and 14).
Interestingly, MMC
rarely occurred in the intestines of the C57BL nude mice which were
used as control mice in the WZWv experiment (Table 9; Appendix).
The high numbers of MMC in the intestines of nude mice, in this
study, might be attributed to a number of factors.
liT-Iineage'' or "T-
Iike" cells have been shown to occur in nude mice
spleen
(Raff,
1973;
Ropke, 1977;
bone marrow and
MacDonald et al., 1981).
The "T-
lineage" cells in nude mice evidently occur in low numbers and are
limited in their functional capacity in comparison to T lymphocytes
from normal mice
(MacDonald,
1984).
Also,
they apparently occur in
higher numbers in older nude mice (MacDonald, 1984).
Nude mice which
developed high numbers of MMC in this study were younger (2-4 months)
than
the
nude
lineage" cells,
mice
which had few MMC
if present at any level,
influence over the mast cell precursor.
(5-10 months),
might have
however,
"T-
exhibited some
The mast cell precursor has
been demonstrated to occur in the intestines of nude mice (Schrader et
al., 1983).
Owen et al. (1975) suggested that the "T-Iineageli cells in nude
mice are of maternal
origin when a heterozygous female is used for
82
breeding.
The demonstration of uT-Iineagei' cells in nude mice from
homozygous
crosses
(Amstutz
et
a l .,
1976)
eliminated
maternal
influence as a sole explanation for the higher than normal numbers of
MMC in nudes in this study.
Alternatively, the explanation for high numbers of MMC in CS7BL
and
BALB/c
nudes
could
be
that
some
element
of the EL diminuta
infection provides an influence for MMC accumlation which functions
independently
possibility,
of
functional
T
lymphocytes.
To
explore
this
CS7BL and BALB/c nudes were inoculated with either H.
diminuta or IL brasiliensis.
C57BL and BALB/c nudes (all sacrificed on
day 12 PI) had MMC in their intestines with both parasites.
It may be that a combination of the parasite
"selectively
stimulus
and
a
functional" T-Iineage cell, which represents a specific
sub-type, caused the development of MMC in the nudes.
Since T cell
subsets are involved in different manifestations of the response to
parasites
(Mitchell, 1979;
Bloom,
1979)
the
presence
of
a T
cell
influence on MMC, with the absence of a T cell influence of suppression
might also be the key to expaining the MMC response in nude mice in
this study.
Reports of higher than normal levels of eosinophils in the
intestines of parasitized nude mice (Andreassen et al., 1978) support
the idea that T suppressor cell activity is a missing part of the nude
T cell repertoire.
A mutation in the nude mouse from the pure nude strain, resulting
in some development of the non-functional reminant thymus, could also
be the cause for the high intestinal MMC observed in this study.
is unlikely
since both colonies would have
had
to
This
simultaneously
83
undergo mutations. . Further, doubt is cast on this possibility by the
fact that no thymus tissue was found among tissue removed from the
mediastinum of nude mice.
The nude status of the BALB/c strain was
established by H-2 and QA gene typing (McLaughlin Research Institute,
Great Falls, Montana) one year prior (1984) to the initiation of this
study.
Eosinophils accumulated in the intestines of C57BL and BALB/c
littermates, and BALB/c nudes in response to H>. diminuta. but did not
accumulate in the intestines of C57BL nudes.
In the BALB/c mice, the
increase in eosinophil numbers occurred in both early and late peaks.
Intestinal eosinophils peaked significantly in the C57BL littermate at
day 12 PI, otherwise remaining at a level constant with the level of
eosinophils in the intestine of C57BL nude mice.
The
lower
eosinophil
numbers in the intestines of nude mice
compared to littermates, especially in the case of the C57BL, might be
attributed to the thymus deficiency.
the action of lymphokines
(Ruitenberg
et
a l . , 1977;
released
Goven,
Eosinophils may be enhanced by
from activated
1983),
but
are
T
lymphocytes
not
completely
dependent upon an intact thymus, since they occurred in nude mice in
response
to
response
to
Yoshimura,
diminuta. and have been reported in nude mice
other
1986).
parasites
(Sugane
arid Oshima,
1982;
in
Ishida and
The levels of intestinal eosinophils in nude mice
were similar or higher than the levels observed in both repaired and
unrepaired WZWv mice.
Elevated levels of peripheral blood eosinophils appeared to occur
in all groups of mice in all experiments except the BALB/c nudes. The
84
occurrence of elevations of blood eosinophils ranged from days 10-17
PI, which was prior to eosinophil accumulation in the small intestine.
Since eosinophils in the blood of parasitized animals reflects
migration of the
cell between tissue
the
compartments, increased levels
should be detectable in the blood before they are
detected
in
the
tissue (Basten et al., 1969)•
Variability in the blood eosinophil response is well documented
in. both parasite infected and non-infected animals
1978; Basten et al., 1979; Sakai,
1981).
(Oglivie et al.,
The sampling of animals at
consistent times each sample day reduces the variability
1974).
(Colley,
Still, considerable variability in blood eosinophilia existed
among and within animal groups.
for one LM mouse
The peripheral blood eosinophil values
sampled over time PI
(of IL_ diminuta inoculation)
provided a striking example of this variability (Fig. 9)•
The failure to detect parasite-specific IgE in the sera of H.
diminuta infected mice is in accordance with previous studies (Befus,
1977)•
It is possible that specific IgE production does occur in LM,
W/Wv and WZWvBM mice, but is so minimal as to not be detectable in the
sera by PCA tests.
the
The use of a more sensitive radioimmunoassay for
detection of IgE may have revealed minimal
(Grammer et al., 1983).
levels in the sera
The failure to detect IgE in the sera of
H.
diminuta infected nude mice was expected, since the production of IgE
has been shown to be clearly dependent upon a functional thymus (Reed
et al., 1982).
The lack of detectable
the
level
of
MMC
parasite-specific IgE is compatable with
accumulation in H.
diminuta infected mice.
The
85
numbers of MMC in LM and WZWvBM mice infected with
brasiliensis and
Zz. spiralis are reported to be 2-5 x higher than the numbers observed
in response to H. diminuta
(Crowle, 1983; Ha et al., 1983).
Parasite-
specific IgE production is also reported to ■be high in response to N.
brasiliensis and
spiralis infections in mice, and to be particularly
high in the case of WZWv mice infected with
brasiliensis (Crowle and
Reed, 1981).
The lower MMC response observed in H l diminuta inoculated LM and
WZWv mice, compared to the MMC response observed with other parasites
may be due to the nature of the tapeworm’s interaction with the host
tissue.
Hl diminuta is a lumen dwelling parasite which has limited
contact with the host intestine.
In constrast, both JCl spiralis and N.
brasiliensis have migratory larval stages which, because of increased
contact with host tissue, may increase the intensity of the cellular
response, especially the eosinophil response
al.,
1981).
(Kay,
1979; Dessein et
The intestinal niche of the adult stage of the parasite
might also influence the intensity of the response; JCl
spiralis is
located intracellularly in enterocytes (Wright, 1979), N. brasiliensis
entwines around the intestinal villi (Kassai,
1982), and H l diminuta
attaches via a scolex to the gut wall (Owen,
1972).
The attachment
area of the tapeworm scolex is limited in comparison to the area of
direct
contact
made
by
comparable
burdens
of
Tl
spiralis or M.
brasiliensis (Gray, 1976; Hasten et al., 1979; Kay, 1979; Issak, 1983;
Reed et al., 1984).
The
source
of
Hl
diminuta
antigens
may be the scolex,
the
tegument, or any number of metabolic or secretory products of the worm.
86
Since the distribution of MMC in the intestines was not isolated to a
single area of parasite attachment, as evidenced by the distribution of
MMC in nearly equal numbers throughout the 56% of sampled intestine
(Sections 1-4, Table 2), the stimulus for MMC accumulation may also not
have been localized to an area of direct contact.
Andreassen et al.
(1978) proposed that the scolex is the primary source of functional
antigen based on the observation that the larger the worm burden (more
scolices) the more rapid the rejection even though total worm mass was
less (5 worm infections vs. 2 worm infections).
If the tegument was
the primary source of antigens, then more tegument (with more biomass)
should induce a stronger host response.
Antigens released from the
tegument of a large tapeworm and processed at the intestinal epithelium
could provide a considerable stimulus for the host response (Befus et
al., 1977).
Many
factors
explusion.
are
involved in the host mechanism of parasite
Synergistic interactions among numerous
components would
result in an optimal host response to the parasite as opposed to the
singular activity of one cell.
the
absence
of
Therefore, rejection of a parasite in
a specific component,
such as the MMC, or delayed
rejection in spite of its presence, does not necessarily diminish the
importance of that
component as a part of the host
response.
The
capacity of the MMC to respond non-speciflealIy to multicellular
antigens which
invade the host
environment indicates that
defense.
at
surfaces exposed to the external
the MMC performs
in
a 'first
line'
of
The fact that this capacity may sometimes be manifest as a
87.
pathological allergic response (Barret and Metcalfe, 1984) adds to the
value of the study of the MMC in the parasite system.
88
SUMMARY
The role of the mucosal mast cell (MMC) in the host response to
parasites
location
remains
controversial
of the MMC at a site
in spite of extensive study.
of parasite
protective role for these cells in the host
invasion,
response
The
indicates
a
to parasites,
which may include involvement in mechanisms of parasite rejection,
regulation of the response,
and repair of parasite damaged tissue.
These suggested roles for the MMC in the host response to intestinal
parasites are based on both the potential biological activities of the
mediators contained within mast
cell granules and
on
the
temporal
association of the mast cell response to parasite rejection (Urquhart
et al., 1965;
Ferguson and Miller,
1979;
Levy and Frondbza, 1983;
Barret and Metcalfe, 1984).
MMC
rejected
responses
(Wells,
1962;
are
usually detectable after the parasite is
Keller,
1971; Mitchell et al., 1983).
The
release of granule components from activated mast cells could render
the MMC unstainable when it is most active.
Therefore, the observation
of increased numbers of MMC in the intestinal villi of the host just
subsequent
to
parasite
rejection may be indicative of the recent
participation of the cell in the rejection.
Increased numbers of MMC
in the intestinal villi well after parasite rejection may be indicative
of alternative roles for the cell
(Ferguson et al., 1979; Askenase,
1980 ).
The use of mast cell deficient W/Wv mice has shown that the MMC
is not essential for the rejection of H l diminuta.
Similar growth and
89
rejection patterns of IL_ diminuta occurred in mast cell deficient W/Wv
mice and mast cell repaired W/Wv mice.
substantial
IL_ diminuta worms grew to a
size in both groups of mice and were
retained
for
an
extended period of time in comparison to normal littermates in which
worms grew to much shorter lengths and were quickly rejected.
The
lack
of
a functional
thymus
in nude mice affects the T
lymphocyte dependent functions of the animal, including the development
of MMC.
Comparison, of the dynamics of
diminuta infections in W/Wv
and nude mice showed that worms generally grew to be longer and heavier
in the W/Wv and were retained for a longer period of time.
of
the
dynamics of
Comparison
diminuta infection in G57BL and BALB/c nude
strains showed a similar pattern of worm rejection from the two nude
strains, but worms grew to a much larger size in the BALB/c nude.
Since the MMC is defined as being dependent upon an intact thymus
for development its occurance in high numbers in the intestines of both
C57BL
and
BALB/c
nudes
was
unexpected.
The high numbers of MMC
observed in the intestines of nude mice in this study may have been due
to the presence of T-Iineage
cells, which occur in the bone marrow,
spleen and blood of nude, mice (MacDonald, 1984) and, if present in the
nude mice in this study, may have induced the development of MMC from
precursor cells in the nude intestines (Schrader et al., 1983).
The
influence of a thymus may not always be essential to the development of
MMC, another stimulus, perhaps from the parasite, might serve to elicit
the response.
The eosinophil responses in the intestines and peripheral blood
were highly variable
both
among
and within
all
groups
of mice.
90
Variability in eosinophilia and higher numbers in LM mice compared to
W/Wv and nude mice may reflect immune deficiencies in the latter two
groups or may be inherent to the eosinophil response. Eosinophils are a
prevalent component, of parasite infections (Oglivie et al., 1978; Kay,
1979) and may help in the rejection of parasites or to modulate the
activity of MMC.
The host response to a parasite stimulus is dependent upon both
the nature of. the host and the nature of the parasite.
diminuta
exists in the lumen of the host intestine in limited direct contact
with the tissue via its scolex.
If functional antigens are primarily
released from the scolex of H jl diminuta. then the cellular and humoral
responses elicited in the host might be less intense than responses
elicited by other intestinal parasites, such as Ni brasilienesis and T.
spiralis, which exist in more intimate contact with the host tissue.
However, functional antigens released from the tapeworm tegument, not
in direct
contact with the host tissue,
could
also
be
active
in
elliciting a host response (Befus et al., 1977),
The production of IgE
W/Wv mice,
may occur in Hi diminuta infected LM and
but was undetectable in the sera by PCA tests.
A more
sensitive test for serum IgE, such as a radioimmunoassay (Crammer et
al., 1983) may have detected minimal levels in the serum.
However, if
a minimal amount of IgE is produced in response to Hi diminuta, then it
would most likely be bound to the MMC in the intestinal mucosa and hot
free in the circulation.
The intermediate host source of Ti molitae or Ti confusum. may
also
influence
the
host
response, however, differences
in
the
definitive host responses might reflect differences in the strain of H.
diminuta used as a source of infective eggs (for the beetles).
The use
of a single source of infective eggs for both intermediate hosts would
help
qualify
the influence of the intermediate host
source on the
definitive host response.
Although the mast cell may not be required, for parasite explusion
from the host, it may still contribute to the rejection process through
int e r a c t i o n s
with
other
components
of
the
host
system.
The
accumulation of MMC in the intestines of Hr diminuta infected LM and
WZWvBM mice in this study indicates that mast cells are involved in the
host response to this parasite.
92
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105
Table 9-
DAY PI
0
6/7
Mean values for worm length, weight, mucosal mast cells
(MMC) per 10 villus crypt units (VCU) and eosinophils (EOS)
per 10 VCU in littermate (LM), W/Wv , W/Wv bone marrow
repaired (WZWvBM), and control C57BL nude (CS7BL NU) mice on
0, 6/7, 10, 12, 16, 21, and 24 post inoculation (PI) with 3
cysticercoids IL_ diminuta. Intermediate host; Tenebrio
molitae. Data in this table are represented in Figs. I, 2, 4,
and 5.
GROUP
12
3
WEIGHT ■
mg
MMC 2
EOS
per• 10 VCU
NP
12.3( 4?
±6.7
12.3( 4)
. ± 7.3
NP
NP
5.3 ( 4)
± 3.9
7.0( 4)
• ± 3.0
NS
NS
LM
NP
w/wv
W/WVBM
NS
NS
LM
3.5(5)
± 3.0
0.9
± 0.7
11.7( 7)
± 7.2
. 41.2(10)
±13.0
w/wv
11.0(6)
± 7.3
3.1
±4.4
3.6( 8)
±4.8
20.8 ( 9)
±8.8
W/WVBM
10.3(5)
± 5.0
1.1
± 3.0
25.0( 5)
±27.5
38.7( 4)
± 7.8
8.9(1)
3.0
4.0 ( 2)
52.5( 2)
47.4( 5)
±19.6
143.4( .5)
±23.6
C57BL NU
10
LENGTH I
cm
• NP
LM
' NP . '
w/wv
63.9(5)
±18.7
7.2
±3.3
1.2 ( 5)
± 1.8
29.8( 4)
± 3.5
C57BL NU
6.2(2)
4.8
1.0( 2)
18.5( 2)
LM
NP
NP
28.8 ( 4)
±14.2
42.3( 4)
± 7.8
w/wv
78.2(5)
±45.5
21.8
±32.4
0.8 ( 5)
± 2.4
41.5( 4)
±13.3
W/WVBM.
69.6(5)
±17.0
21.8
± 9.9
18.8( 4)
±23.1
50.3( 4)
±13.5
Table 9 (continued)
DAY PI
16
21
24
GROUP
LM
LENGTH
cm
WEIGHT
mg
MMC
NP
■ NP
47.7(9)
±28.0
42.7(9)
±15.5
EOS
per 10 VCU
w/wv
93.0(4)
±50.9
60.8
±54.5
3.0(9)
± 3.1
41.9(7)
±14.2
W/WVBM
100.0(1)
56.0
40.2(5)
. ± 9.8
48.0(5)
± 5.8
C57BL
48.1(1)
21.0
6.0(2)
19.0(2)
NP
94.0(8)
±31.7
. 126.9(7)
±60.5
LM
NP
W/WV
82.4(3)
61.0
10.4(7)
± 5.0
58.8(7)
±22.7
WZWvBM
58.8(1)
27.0
55.0(5)
±28.2
73.3(3)
± 2.9
C57BL
NP
NP
3.5(2)
90.5(2)
LM
NP
NP
7.0(3)
±2.6
43.7(3)
±26.6 .
• w/wv
NP
NP
2.5(2)
20.5(2)
NS
NS,
NS
WZWvBM
NS
1. length and weight values are means of all worms per positive mouse
2. mucosal mast cells (MMC) and eosinophils (EOS) per 10 villus crypt
units values are means of counts from all mice killed on a given
sample day.
3. NP = no positives (no mice positive for worms), NS = no sample.
4. parenthesis enclose number of mice included in count.
Table 10.
Eosinophils per cubic millimeter of peripheral blood collected from littermate
(LM), W/Wv , W/WV bone marrow repaired (W/W BH), and control C57BL nude (C57BL
NO) mice on alternate or specific days post-inoculation (PI) with 3 Cysticerddids
of H. diminuta ± SD. Data from this table are represented in Fig. 6.
GROUP
DAY PI
-I
I
3
5
7
9
11
13
15
17
19
21
560
±285
290
±204
730
±515
540
±319
430
±120
490
±170
930
±329
1180
±329
1010
±402
1010
±315
410
±182
560
±290
w/wv
290
±213
230
± 57
430
±148
340
±150
380
±130
230
±214
160
± 55
380
± 91
740
±299
740
1270
±965 .±414
360
±188
W/WVBM
450
±325
220
±175
600
±419
560
±135
670
±135
450
±234
500
±258
860
±438
1530
±164
1450
±754
870
±333
620
±289
C57BL NU
550
100
930
550
107
LM
108
Table 11.
DAY PI
6
12
Mean values for worm length, weight, mucosal mast cells
(MMC) per 10 villus crypt units (VCU) and eosinophils (EOS)
per 10 VCU in littermate (LM) and W/Wv mice on days 6, 12,
18, and 24 post-inoculation (PI) with 3 cysticercoids of h.
diminuta Intermediate host: Tribolium confusum. Data from
this table are represented in Figs. 8, 9> and 10.
LENGHTi
cm
-WEIGHT
■ mg
LM
N.P3
NP
38.8(4)4
±23.4
78.0(3)
± 5.0
W/Wv
n ;p
NP
10.5(2)
± 5.0
46.7(3)
±14.0
LM
NP
NP
64.8(4)
±29.4
80.3(3)
±76.0
8.6(3).
± 3.8
32.0(5)
±16.6
48.0(3)
±30.5 .
73.4(5)
±32.8
GROUP
W/Wv
18
LM
W/Wv
24
88.7(4)
±36.0
N .
63.3(2)
± 2.8
33.8(4)
±34.1
N
EOS
MMC 2
per 10 VCU
5.0(4)
60.0(2)
. ±10.0
±14.1
34.0(4)
±20.4
LM
NP
NP
96.4(5)
±19.6
144.0(5)
±55.1
W/WV.
NP
NP
4.0(5)
± 4.4
108.0(3)
± 6.4
I. length and weight values are means of all worms in all worm
positive mice
2. mucosal mast cells (MMC) and eosinophils (EOS) per 10 villus
crypt units (VCU) are means of counts from all mice killed
. on a given day.
3. NP = no positives (no mice positive for worms)
4. parenthesis enclose number of mice included in count.
I
109
Table 12.
DAY PI
0
7
12
Itean values for worm length, weight, mucosal mast cells
(MMC) per. 10 villus crypt units (VCU) and eosinophils (EOS)
per 10 VCU in C57BL littermate (LM) and nude (NU) mice on
days 0, 7, 12, 16, and 21 post inoculation (Pl) with 3
cysticercoids of Hr diminuta.
C57BL LM
NP3
NP
52.7(3)4
±26.5
46.7(3)
± 3.1
C57BL NU
NP
NP
42.0(3)
±12.8
43.7(3)
±6.1.
C57BL LM
2.4(3)
± 0.9
0.5
±0.0
46.3(3)
±15.8
93.7(3)
±39.6
C57BL NU
5.7(2)
± 6.5
0.5
±0.0
33.0(3)
± 9.6
104.3(3)
±29.1
NP
128.0(3)
±34.4
224.0(3)
±12.1
6.0
±6.0
42.0(3)
± 8.6
64.3(3)
±20.2
NP
131.0(3)
±48.1
67.7(3)
±31.6
4.0
±2.0
58.7(3)
• ±13.6
69.0(3)
±24.6
NP
150.7(3)
±4.8
31.3(3)
±14,5
2.0
75.0(3)
±32.5
25.5(3)
± 9.2
.C57BL LM
C57BL.LM
C57BL NU
21
"
WEIGHT
mg
C57BL NU
16
EOS
MMC2
per 10 VCU
LENGTH 1
cm
GROUP
C57BL LM
C57BL NU
NE
28.8(3)
±13.3
NP
37.1(2)
±45.9
NP
'
29.9(1)
1. length and weight values are means of all worms in all positive
mice.
2. mucosal mast cells (MMC) and eosinophils (EOS) per 10 villus
crypt units (VCU) are means of counts from all mice killed on
a given sample day.
3. NP = no positives (no mice positive for worms).
4. parenthesis enclose number of mice included in count=
HO
Table 13.
DAY PI
0
7
12
Mean values for worm length, weight, mucosal mast cells
(MMC) per 10 villus crypt units (VCU) and eosinophils (EOS)
per 10 VCU in BALB/c littermate (LM) and nude (NU) mice on
days 0, 7, 12, 16, and 21 post-inoculation (PI) with 3
cysticercoids of Hjl diminuta.
GROUP
LENGTH 1
cm
WEIGHT
mg
BALB/c LM
N-F34
NP
38.O O ^
±24.0
63.7(3)
± 7.4
BALB/c NU
NP
NP
28.0(3)
±12.8
64.3(3)
± 9.3
51.7(3)
±20.3
130.0(3)
±23.1
BALB/c LM
.
NP
NP
57.0(3)
±15.0
81.5(3)
±27.6
BALB/c LM
NP
N P
87.7(3)
±21.4
50.3(3)
±22.2
9.7
±10.0
50.0(3)
±12.4
49.3(3)
±15.9
N P
164.0(3)
±23.5
81.0(3)
±47.1
7.0
58.0(3)
±12.1
73.3(3)
±26.0
BALBzC LM
BALB/c NU
21
1.0
BALB/c NU
BAL^c NU
16
13.4(1)
MM C 2
EOS
per 10 VCU
38.8(3)
±25.1
NP
111.2(1)
BALBzC LM
NP
N P
97.0(3)
±14.8
22.3(3)
± 2.1
BALBzC NU
N P
NP
32.7(3)
± 5.5
20.3(3)
±11.1
I. length and weight values are means of all worms in all worm
positive mice.
2. mucosal mast cells (MMC) and eosinophils (EOS) per 10 villus
crypt units (VCU) are means of counts from all mice killed on
a given sample day.
3. NP = no positives (no mice positive for worms).
4. parenthesis enclose number of mice included in count.
Eosinophils per cubic millimeter of peripheral blood collected from C57BL
littermate (C57BL LM), C57BL nude (657BL NU), BALB/c littermate (BALB/c LM)
and BALB/c nude (BALB/c LM) mice on alternate days post-inoculation (PI)
with 3 cysticercoids of
diminuta + SD. Data from this table are
represented in Figs. 17 and 18.
GROUP
DAY PI
0
2
4
6
8
10
12
14
16
18
20
C57BL LM
130
± 60
230
±130
500
±180
260
±150
300
±160
980
±401
520
±260
600
±500
370
±180
500
±250
410
±300
C57BL NU
250
±150
500
±140
460
±160
220
±220
310
±108
230
± 44
740
±170
450
±170
1140
±750
570
±180
280
±300
BALB/c LM
470
±100
400
340
±190. ±180
210
± 85
450
±260
630
±500
560
± 21
790
±450
320
± 71
280
±175
350
±250 •
BALB/c NU
165
±125
190
±150
370
± 50
250
± 35
180
± 65
180
± 25
210
± 50
300
±100
200
± 85
320
± 75
380
±250
TtT
Table 14 .
MONTANA STATE UNIVERSITY LIBRARIES
CP
65
CO
CM
O
O
f
CM
CD
I—
CO
11111111111I anIII