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T Cell-Deficient Mice Exhibit Reduced Disease Severity and Decreased Inflammatory Response in the Brain in Murine Neurocysticercosis
Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
and a type 1 pathway of cytokine expression. To understand the role of
T cells during this infection, the cellular and cytokine
response was analyzed in mice that lack T cells
(TCR-/-). In TCR-/- mice,
Mesocestoides corti metacestodes preferentially invaded
the extraparenchymal areas of the brain. Furthermore, parasites were
able to escape from the brain and establish a systemic infection with
liver and peritoneal involvement. Immunopathological studies indicated
that TCR-/- mice develop little inflammatory response
and less neurological symptomatology. Significantly reduced numbers of
T cells, macrophages, dendritic cells, and mast cells were present in
the brain. The cytokine response in the brain of
TCR-/- mice appears to be a mixed type1/type 2
response with low levels of IL-2, IL-4, IL-10, IL-12, IL-13, IL-15, and
IFN-. To further investigate the immunological significance of this
cell population, T cells were adoptively transferred into
intracranially infected TCR-/- mice. T cells
were specifically recruited into the CNS in response to this parasitic
infection, and they were able to target the infected brain within
12 h after transfer. These results suggest that T cells are
key players in the immune response elicited during this CNS infection
and direct a type 1 response in wild-type mice upon
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the mouse model for NCC, mice are i.c. inoculated with
Mesocestoides corti metacestodes (8). In
wild-type mice, CNS infection is characterized by a massive influx of
T cells, as determined by in situ immunohistochemistry of brain
cryosections (8). In addition, the brain immune response
is associated with a predominant type 1 pathway of cytokine response.
The T cell response is induced very rapidly after infection
(2–3 days postinfection (p.i.)) and predominates during the course of
the infection. This finding is of interest because T cells
represent a minor T cell population residing in lymphoid organs
(9, 10). In contrast, T cells are a predominant T
cell population within epithelial tissues such as the skin, intestine,
and lung (9, 10, 11, 12). T cells have been demonstrated to
actively participate in host immune response by regulating and
resolving inflammatory processes (13, 14, 15). Circulating
T cells have been found to increase in several infectious states
(16) such as tuberculosis, malaria, toxoplasmosis, and
HIV. An early protective role of T cells during Listeria
monocytogenes infection has been demonstrated (17).
T cells have also been implicated in controlling HSV replication
and spread, through potent cytotoxic responses (18). The
contribution of T cells in the initiation of inflammatory
response during influenza virus infection suggests a role for T
cells in viral immunity (14, 19). Furthermore, T
cells appear to modulate host immune response in several pathologies by
producing several type 1 and/or type 2 cytokines (20, 21),
including IFN-, IL-2, IL-4, and IL-10 (22, 23).
However, under pathological conditions, T cells have not been
described as a major lymphocyte population in the CNS.
To investigate the role of T cells during this infection, we
characterized the CNS immune response to M. corti in TCR
-chain-/- mice
(TCR-/-) that lack T cells. The
studies include an analysis of the pathology, cell types, and cytokine
expression of infiltrating leukocytes, migratory patterns of the
parasite, and cell trafficking experiments upon adoptive transfer of
T cells into TCR-/- mice.
The results indicate that T cells are critical immunoregulatory
players in the infection and further define the immune function of this
T cell subset, including their potential role in the human disease of
neurocysticercosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice 3–5 wk old were purchased from the National
Cancer Institute Animal Program (Bethesda, MD). Female C57BL/6 and
-chain TCR-/- mice
(TCR-/-) were purchased from The Jackson
Laboratory (Bar Harbor, ME). Animal experiments were conducted under
the guidelines of the University of Texas System, The U.S. Department
of Agriculture, and the National Institutes of Health.
Parasites and inoculations
M. corti metacestodes were maintained by serial i.p. inoculation of 8- to 12-wk-old female BALB/c mice (8). Intracranial inoculations were performed, as described previously (8). Control mice were injected with 60 µl sterile HBSS using the same protocol. Before i.c. inoculation, mice were anesthetized i.m. with 20 µl mouse mixture containing 100 mg/ml ketamine and 20 mg/ml rompum (Laboratory Animal Resource, University of Texas Health Science Center, San Antonio, TX). Animals were sacrificed at several time points after inoculation and analyzed for parasite burden and various immune parameters. Before sacrifice, animals were anesthetized with 100 µl mouse mixture and perfused through the left ventricle with 10 ml cold PBS.
Tissue processing
The brain was immediately removed from perfused animals, embedded in O.C.T. resin (optimal cutting temperature), and snap frozen. Serial horizontal cryosections 10 µm in thickness were placed on silane prep slides (Sigma-Aldrich, St. Louis, MO). One in every four slides was fixed in Formalin for 12 min at room temperature and stained with H&E. The remainder of the slides was air dried overnight and fixed in fresh acetone for 20 s at room temperature. Acetone-fixed sections were wrapped in aluminum foil and stored at -80°C or processed immediately for immunohistochemistry or immunofluorescence.
H&E staining
After fixation in 4% Formalin for 10 min at room temperature, slides were washed twice in deionized water, dehydrated 30 s in 100% ethanol, stained 30 s in hematoxylin, and washed in distilled water for 2 min. Tissue sections were stained with eosin for 15 s, followed by 2-min treatment with 95 and 100% ethanol each. Slides were allowed to air dry, submerged in xylene for 3 min, and mounted using cytoseal mounting medium (Stephens Scientific, Riverdale, NJ). The number and location of parasites were determined by microscopic examination of the stained tissues. Tissues were also analyzed for the presence or absence of mononuclear infiltrates.
Antibodies
Abs purchased from BD PharMingen (San Diego, CA) include GL3
(pan anti-), H57-597 (pan anti-
), 5E6
(anti-NK), M1/70 (anti-Mac1), Cy34.1 (anti-CD22), XMG1.2
(anti-IFN-), SXC-1 (anti-IL-10), BVD6-24G2 (anti-IL-4),
JES6-5H4 (anti-IL-2), G277-3960 (anti-IL-15), RB6-8C5
(anti-Gr1), and 9A5 (anti-IL-12 heterodimeric p70). The
purified anti-mouse polyclonal Ab against IL-13 was purchased from
R&D Systems (Minneapolis, MN). The purified Ab, NLDC-145
(anti-DEC205 Ab specific for dendritic cells and thymic epithelial
cells), was obtained from Serotec (Raleigh, NC). All Abs were titrated
in spleen sections from i.p. infected mice to determine optimal
concentrations. Spleen sections were used as positive controls in all
experiments. Mast cells were detected using toluidine blue
staining.
Brain mononuclear cell isolation
Mice were sacrificed 7 days after adoptive transfer. Each perfused brain was gently minced through a fine 150-µm Nitex screen (Sefar America, Depew, NY) using a syringe plunger and collected into 10 ml HBSS (Invitrogen, Carlsbad, CA) containing 0.05% collagenase D (Roche Diagnostics, Indianapolis, IN), 0.1 µg/ml L-1-chloro-3[4-tosyl-amido]-7-amino-2-heptanone-HCl (Sigma-Aldrich), 10 mg/ml DNaseI (Sigma-Aldrich), and 10 mM HEPES buffer, pH 7.4 (Invitrogen). The mix was gently rocked at room temperature for 1 h and allowed to settle at unit gravity for 30 min to deplete undigested debris. Supernatant was collected and pelleted at 200 x g for 5 min and resuspended in 3 ml Ca2+/Mg2+-free HBSS (Invitrogen) for each brain. Cells were washed three times in 1 ml 0.1% BSA (Sigma-Aldrich) in HBSS and counted. Cells were diluted to 2 x 105 cells/ml, and a 0.5-ml aliquot was cytocentrifuged in a Cytospin 2 (Shandon, Pittsburgh, PA) onto silane prep slides (Sigma-Aldrich).
Peritoneal leukocyte isolation
Intraperitoneal leukocytes were isolated from C57BL/6 mice that
were infected i.p. for 3–5 wk with M. corti or from normal
uninfected mice. The peritoneal cavity was washed with 10 ml HBSS, and
the preparation was filtered through a 150-µm nylon screen (Sefar
America) to remove parasites. Cells were pelleted at 300 x
g for 10 min and washed three times in 0.1% BSA in PBS and
counted. For immunocytochemistry, cells were diluted to 2 x
105 cells/ml in 0.1% BSA, and 0.5 ml of this
cellular suspension was cytocentrifuged into "super plus"
covered slides (VWR Scientific Products, Sugar Land, TX) at 1000 rpm
for 7 min. For positive selection of T lymphocytes, the cell
suspension was incubated with 10 µg/ml Dynabeads M-280 streptavidin
(Dynal, Lake Success, NJ) previously coated with 500 µg/ml
biotinylated GL3 Ab (BD PharMingen) for 20 min at 4°C. Cells were
then selected using a magnetic particle concentrator (Dynal) and washed
eight times, according to manufacturer’s instructions.
CSFE labeling
CFSE (Molecular Probes, Eugene, OR) was diluted to 5 mM in DMSO
(Sigma-Aldrich), aliquoted, and stored at -20°C, protected from
light under desiccating conditions. Cells were resuspended at 5 x
107 cells/ml in prewarmed RPMI 1640 (Invitrogen)
supplemented with 20 mM HEPES, 0.01 M sodium bicarbonate (Invitrogen),
0.05 mg/ml penicillin G (Invitrogen), 0.1 mg/ml streptomycin
(Invitrogen), and 10% FCS (Sigma-Aldrich). This medium will be
subsequently referred to as RPMI-10. A total of 10 µl 5 µM CFSE
diluted in RPMI-10 was added to the cells and incubated at 37°C with
inversion every 3 min. To stop labeling, several volumes of ice-cold
RPMI-10 were added, and cells were washed twice in RPMI-10. Cells were
then resuspended to 5 x 106 cells/ml when
positively selected T cells were labeled or 2 x
107 cells/ml for total peritoneal lymphocytes.
CFSE-labeled cells were immediately injected into recipient
mice.
Adoptive transfer of T cells into TCR-/-
mice
An adoptive transfer experiment was designed with six animals
per group, in which three brains were processed for cryostat sectioning
and three for isolation of brain leukocytes for analysis by
immunocytochemistry. Recipients were TCR-/-
mice i.c. infected with M. corti for 4 wk (i.c.
TCR-/-) or injected i.c. with HBSS (HBSS
TCR-/-). Two populations of CFSE-labeled
donor cells were used: 1) total peritoneal leukocytes from i.p.
infected C57BL/6 mice referred to as infected cells, and 2) total
peritoneal leukocytes from normal C57BL/6 mice (normal cells).
Experimental groups are described as follows: group I, i.c.
TCR-/- transferred with infected cells;
group II, i.c. TCR-/- transferred with
normal cells; group III, HBSS TCR-/-
transferred with infected cells. Control animals included i.c. infected
TCR-/- mice and HBSS
TCR-/- mice i.v. injected with 200 µl
RPMI-10. Tissues were analyzed for the presence and distribution of
infiltrating cells. CFSE-labeled lymphocytes (green fluorescence) were
detected by direct microscopic examination of brain sections under a
fluorescent microscope using the FITC filter. Immunofluorescence
performed with the biotinylated GL3 Ab followed with the
streptavidin-Alexa fluor 568 conjugate (red fluorescence) allowed the
visualization of dual-labeled T cells as yellow fluorescence.
The proportion of T cells, T cells, and Mac-1-positive
cells was obtained after immunocytochemistry of isolated brain
leukocytes 3 and 7 days after adoptive transfer.
In a separate experiment, T cells were first isolated from
peritoneal lymphocytes of i.p. infected C57BL/6 mice and then labeled
with CFSE before injection into recipients. Three
TCR-/- recipient mice i.c. infected with
M. corti were injected with 1 x
106 CFSE-labeled T cells in 200 µl, and
brain cryosections were analyzed after 12 h for the presence and
distribution of T cells, as described above.
Immunohistochemistry
Brain tissue sections or cytocentrifuged brain leukocytes were stained with specific biotinylated Abs, as described previously (8). For detection of IL-12, sections were incubated with purified anti-IL-12 Ab, followed by incubation with biotinylated goat anti-rat IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Both Abs were diluted in 3% BSA and incubated for 30 min at 37°C. Tissues were then incubated with streptavidin-alkaline phosphatase for 30 min at room temperature and developed using the alkaline phosphatase substrate kit I (Vector Laboratories, Burlingame, CA). In between incubations, tissues/cells were washed three times for 3 min each. Similarly, the biotinylated Ab rat anti-goat IgG was used for detection of anti-IL-13 and anti-DEC205 Abs.
A semiquantitative analysis of the cellular infiltrates was done by counting the number of cells of a given cell type/cytokine per section. All markers were tested on at least three animals. Positive cells were counted, and results were scored as: +, 1–100 positive cells per section; ++, 100–300; +++, 300–500; and ++++, >500. After staining of cytocentrifuged leukocytes, the proportion of positive cells was obtained by counting the number of positive lymphocytes in a total of 1000 cells using x600 magnification.
Immunofluorescence
All incubations were conducted at room temperature and, between incubations, slides were washed six times for 5 min each in 0.1% BSA in PBS. Tissue sections were incubated with the biotinylated GL3 for 1 h, followed by incubation with streptavidin-fluor 568 conjugate (Molecular Probes) for 30 min. Sections were washed and mounted using fluorsave reagent (Calbiochem, La Jolla, CA) containing 0.3 µM 4',6'-diamidino-2-phenylindole dilactate (Molecular Probes).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
Serial horizontal sections of infected mouse brain were stained
with H&E to determine the number and location of M. corti
metacestodes. The associated immunopathology was followed for 13 wk
after infection. In BALB/c and C57BL/6 mice, parasites invaded brain
parenchyma in a time-dependent manner. During the first 5 days after
infection, most of the organisms remained outside the brain. By 1 wk
p.i., the majority of the parasites were located in extraparenchymal
regions, which include meninges, subarachnoid spaces, and ventricles.
By 3 wk p.i., approximately one-half of the organisms had penetrated
brain parenchyma (Table I
, Fig. 1a). In contrast, in mice
lacking T cells, parasites preferentially invaded
extraparenchymal areas of the brain, particularly subarachnoid spaces
(Fig. 1b). At each time point analyzed (1, 3, 5, 8, and 13
wk p.i.), fewer parenchymal parasites were consistently found in
TCR-/- mice compared with wild-type mice
(Table I). In the 13-wk period analyzed, nearly 60% of the parasites
were located in brain parenchyma in wild-type mice, compared with 26%
in TCR-/- mice.
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-/- mice develop fewer neurological
manifestations
CNS infection with M. corti metacestodes in C57BL/6 and
BALB/c mice was associated with the presence of severe neurological
symptoms. Usually during the first 1–3 wk p.i., mice exhibited
symptoms such as staggering, abnormal vestibular functions, including
abnormal landing foot splay, repetitive walking in circles, and weight
loss. With an inoculum of 40 parasites, wild-type mice typically did
not survive longer than 13 wk after infection. During that period of
time, 
60% of M. corti-infected mice (14 of 25 C57BL/6
mice and 19 of 31 BALB/c mice) exhibited intense neurological
manifestations. In contrast, in TCR-/- mice,
symptoms were only detected after 8 wk p.i. and were not as severe as
those observed in wild-type mice even at 13 wk. During the course of
the infection, 30% (11 of 38) of the
TCR-/- mice exhibited an extremely quiet
state. This might be explained by the presence of large number of
parasites in subarachnoid spaces perhaps causing increased i.c.
pressure. However, TCR-/- mice with an
inoculum of 40 parasites typically survived for 20 wk or more after
M. corti infection, a substantially longer period than the
13 wk for normal background mice.
Reduced CNS pathology after M. corti infection
in TCR-/- mice
H&E-stained sections of infected brains were also evaluated to
determine changes in nervous tissue integrity (Fig. 2). Fig. 2a represents brain
tissue from control animals that were i.c. inoculated with HBSS. In
wild-type mice, areas of active necrosis were identified by the lack of
cellularity in parenchymal tissue (Fig. 2b). Areas with
spongiosis were initially detected within the first 2 wk p.i. and were
present throughout the infection. These areas were determined by the
presence of cell drop in areas close to the parasite (Fig. 2c). In TCR-/- mice, areas of
necrosis in brain parenchyma were not detected. However, some of the
parasites located in brain parenchyma were associated with small
regions in which spongiosis was present (Fig. 2d), but to a
lesser extent than wild-type infected mice.
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-/- mice exhibit a systemic disseminated
infection
One of the most interesting observations during the infection of
i.c. inoculated TCR-/- mice was the
detection of parasites outside the CNS. During the first 7 wk after
i.c. infection, 7% (2 of 14) of the
TCR-/- mice revealed the presence of
parasites in liver parenchyma that were detected macroscopically during
the perfusion procedure. By 8 wk p.i., 40% (11 of 24) of i.c. infected
mice displayed a disseminated infection with encystment of the liver
and a massive peritoneal infection. In normal background mice, the
infection was maintained in the CNS. Parasites were not detected in the
liver or peritoneal cavity. These observations indicate that T
cells are critical in maintaining a localized CNS infection.
Decreased abundance of immune cells in the brain of
TCR-/- mice
The immune response in the CNS after infection with M.
corti was analyzed by in situ immunohistochemistry (IHC). The
infection in wild-type mice was associated with a strong inflammatory
response in both extraparenchymal (Fig. 3a) and parenchymal (data not
shown) areas of the brain. In wild-type mice, T cells and
macrophages appear associated with extraparenchymal parasites as early
as 2 days p.i., and the numbers increase dramatically after 1 wk p.i.
(Fig. 3a). Interestingly, T cells constitute the
predominant T cell population during the course of infection (Fig. 3, a and b). T cells (Fig. 3b) and
macrophages were initially found in meninges and subarachnoid spaces.
As the immune response progresses, a large number of cells accumulates
in ventricles. T cells, B cells/plasma cells (Fig. 3a), and dendritic cells (Fig. 3c) were also
found in extraparenchymal infiltrates, although in less abundance. In
contrast to the prominent cellular response elicited by the infection
in wild-type mice, TCR-/- mice developed a
less intense inflammatory response (Fig. 3d). Macrophages
were detected by 2 days p.i. (Fig. 3d) and constituted the
major leukocyte population in the brain of T cell-deficient mice
(Fig. 3e). Moreover, TCR-/- mice
exhibited less accumulation of T cells, B cells/plasma cells,
and dendritic cells (Fig. 3f) within extraparenchymal
infiltrates.
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T cell-deficient mice was delayed and less
prominent (data not shown). In wild-type mice, T cells and
macrophages were consistently present and associated with parenchymal
parasites by 1 wk p.i. In both wild-type and
TCR-/- mice, T cells and B
cells/plasma cells were found associated with parenchymal parasites
after 1 and 5 wk, respectively. As described previously, dendritic
cells were detected in ventricles and subarachnoid spaces in wild-type
(Fig. 3
c) and TCR-/- mice (Fig. 3f). However, in wild-type mice, dendritic cells rapidly
mobilized toward brain parenchyma within 1 wk p.i. In contrast, in
TCR-/- mice, few dendritic cells were
present in parenchymal tissue by 3–5 wk p.i. and were not directly
associated with parasites. In both wild-type and T
cell-deficient mice, polymorphonuclear cells were found in very low
abundance (less than 15 Gr-1-positive cells/tissue section) in both
extraparenchymal and parenchymal infiltrates.
Toluidine blue staining of infected brain sections was used to
determine the presence and distribution of mast cells (Fig. 4). In wild-type mice, mast cells were
detected in extraparenchymal infiltrates (Fig. 4a), both
closely associated to the parasites as well as scattered in brain
parenchyma (Fig. 4b). In TCR-/-
mice, fewer mast cells were detected (Fig. 4, c and
d). In wild-type mice, mast cells were often found in a
degranulated stage.
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-/- mice after M. corti infection
To further characterize the immune response in the brain, the
expression of several cytokines was analyzed by IHC. In wild-type mice,
the infection was associated with high expression of IL-2, IL-12,
IL-15, and IFN- (Fig. 5a).
These cytokines were induced during the first week after infection, and
colocalized to the areas in which T cells were present.
Relatively low expression of the type 2-related cytokines IL-4 and
IL-13 was detected (Fig. 5b), and IL-10 was not detected.
The observed pattern of cytokine expression indicates the predominance
of a type 1 pathway of cellular response in the CNS after M.
corti infection. The cytokine response in the brain of
TCR-/- mice reveals the presence of low
expression of IL-2, IL-12, IL-15, IFN- (Fig. 5c), IL-4,
and IL-13 (Fig. 5d). These results indicate that whereas in
wild-type mice there is a predominant type 1 pathway of cytokine
response, in TCR-/- mice the numbers of
cells expressing type 1 cytokines are reduced and their appearance
delayed. As a result, there is almost an equivalent number of cells
expressing type 1 and type 2 cytokines at 1–13 wk p.i. Using double
immunofluorescence, it was demonstrated in wild-type mice that T
cells produce IL-12 and IFN- (Cardona et al., submitted for
publication).3
Therefore, T cells appear instrumental in the development of a
cell-mediated inflammatory response in the CNS.
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T cells into TCR-/-
mice are found in the inflammatory infiltrates in infected brains
To further examine the role of T cells, we analyzed their
migration pattern and distribution in the brain of
TCR-/- mice. Cells were labeled with the
vital fluorescent dye CFSE. It was hypothesized that the presence of
T cells in the brain of TCR-/- mice
would be able to cause an increase in the numbers of immune cells and,
more importantly, to up-regulate the type 1 inflammatory response. In
initial experiments, isolated T cells obtained from the brains
of i.c. infected C57BL/6 mice were used as donor cells. It was found
that by 12 h after adoptive transfer, T cells were
specifically recruited into the brain of
TCR-/- mice, as detected by the presence of
green fluorescent groups of infiltrating CFSE-labeled T cells in
ventricles and subarachnoid spaces (Fig. 6a). Even though T
cells were found in high numbers in the brain of wild-type i.c.
infected mice, it was difficult to isolate the numbers of positive
selected cells required for the different groups for adoptive transfer
experiment. Moreover, isolation of large numbers of T cells from
normal uninfected mice was not possible.
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T cells
constituted 10–20% of the total peritoneal population of i.p.
infected mice. Moreover, the chemokine receptors expressed by T
cells in the peritoneum of i.p. infected mice were identical with brain
T cells (A. E. Cardona, W. A. Kuziel, and J. M.
Teale, manuscript in preparation). Therefore, in subsequent
experiments, total peritoneal cells that were CFSE labeled were used
instead of brain T cells. The migration of T cells was
analyzed at 12 h, 3 and 7 days after adoptive transfer. In
addition to the CFSE labeling of adoptively transferred cells, brain
sections from recipients were also stained with red fluorescent
anti- TCR so that T cells could be specifically
identified. T cells were found in the brain 3 days after
transfer (Fig. 6, b and c), as detected by the
presence of dual-labeled cells (CFSE labeled, and anti- TCR
positive). It was clear that T cells were able to proliferate in
infected brains, as seen by the intensity of the dual-labeled cells
ranging from bright yellow to orange, indicating loss of the green
fluorescence. In addition, some T cells at this stage lost the
CFSE dye entirely and were observed as red fluorescent cells (Fig. 6b, arrowhead). However, a population of CFSE-labeled cells
was still present in the brain as analyzed at 3 days after transfer
(Fig. 6c).
At 7 days, it was determined whether the transfer of T cells in
TCR-/- mice affected the cell types that
infiltrated the CNS. The proportion of specific cell types was
determined by immunocytochemistry on isolated brain leukocytes (Table II). The specific recruitment of cells
appeared to require infection of recipients with M. corti.
Recipient TCR-/- mice inoculated with HBSS
did not reveal the presence of T cells when donor peritoneal
cells from infected mice were injected (Table II). In contrast,
transfer of lymphocytes from infected mice into i.c. infected
TCR-/- mice caused a significant increase in
the number of infiltrating cells into the brain (Table II). T
cells accounted for 2% of the brain lymphocytes. Furthermore, four
times more T cells and twice as many Mac-1-positive cells were
found after adoptive transfer. In contrast, when
TCR-/- mice were transferred with peritoneal
cells from uninfected mice, no statistically significant changes were
detected in the abundance of T cells and macrophages (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice indicates that T cells
are instrumental immunoregulatory cells that promote and enhance host
immune responses. The cellular inflammatory type 1 response that
predominates in wild-type mice appears mediated by cytokines such as
IL-12 and IFN- that are released early during the infection by
T cells.3 In sharp contrast,
TCR-/- mice exhibited little inflammatory
response, and a mixed type 1/type 2 response was associated with the
infection. Furthermore, T cell-deficient mice exhibited a less
severe disease. The present studies strongly support the influence of
T cells on inflammation and a direct effect of the host CNS
immune response in the pathogenesis of the disease.
The importance of T cells promoting a CNS inflammatory response
was further demonstrated by adoptive transfer of CFSE-labeled T
cells into TCR-/- mice. Transferred T
cells were found in the brains of recipients as early as 12 h p.i.
By 3 days p.i., the numbers of T cells increased, and it was
clear that the transferred T cells were proliferating based upon
their fluorescence profile. Importantly, the presence of T cells
in the brain of mutant mice resulted in an increase of infiltrating
leukocytes by 7 days p.i. These results provided strong support for the
role T cells play in amplifying the immune response, presumably
by their production of crucial cytokines such as IFN- and IL-12. In
addition, T cells were demonstrated to produce several CC
chemokines early after M. corti
infection,4 contributing to the
recruitment of inflammatory cells into the brain. Interestingly, the
transferred T cells appeared to preferentially target the brain
of infected mutant mice, as T cells were found in very low
numbers (5–20 cells/section) in the spleen of recipients and were
localized to marginal areas of the germinal centers. Moreover, the
number of splenic T cells in recipient mice was essentially the
same for all experimental groups. Such findings correlate with our
previous studies in that when normal mice are i.p. infected with the
same organism, the T cell response in spleen and liver is much
less predominant than that found in the brain. Thus, it is possible
that T cells are important immune cells for the brain similar to
their importance in various mucosal tissues.
T cells also affected the infiltration of dendritic cells and
mast cells into the infected brain. Thus, lower numbers of both of
these cell types were found in TCR-/- mice
infected with M. corti compared with normal background mice.
In addition, the mutant mice exhibited fewer dendritic cells in
parenchymal tissue. This is of interest in that dendritic cells are
usually found in the meninges, choroids plexus, and cerebrospinal fluid
(CSF), but not in the parenchyma, unless there is inflammation
(24, 25, 26, 27), again confirming a reduced inflammatory state in
the absence of T cells. In the normal CNS, mast cells have been
identified in meninges, thalamus, and periventricular regions, and
generally localized adjacent to blood vessels (28, 29).
However, the increased numbers of mast cells during neurophatological
conditions and studies from experimental allergic encephalomyelitis
(EAE) model suggest that mast cells play a role in
neurologic inflammation (30, 31). This correlates with the
findings shown in this study in that infected
TCR-/- mice exhibited fewer mast cells and
reduced inflammation and pathology. Mast cells are known to release
mediators that cause tissue edema and extracellular matrix degradation
(32, 33) and are most likely involved in CNS pathology.
Interestingly, the increase in IL-4 and IL-13 relative to the type 1
cytokine in TCR-/- mice would have predicted
a larger mast cell infiltration typical of type 2 responses in contrast
to the decreased mast cell accumulation found. However, the
infiltration of dendritic cells and mast cells into the brain is
associated with several CC chemokines. As T cell-deficient mice
exhibited decreased CC chemokine expression in the brain after M.
corti infection,4 the decreased cellular
infiltration may well reflect chemokine deficiencies.
The differential pattern of parasite distribution in wild-type and
T cell-deficient is not completely understood. M.
corti metacestodes most likely release proteolytic enzymes as a
mechanism to penetrate brain parenchyma. Although hypothetical, it is
possible that the immune response plays a critical role in enhancing
the invasion process and parasite migration toward brain parenchyma by
damaging the peripheral tissue of the brain and further breakdown of
the blood brain barrier. In the absence of a strong inflammatory
response as exhibited in TCR-/- mice,
parasites might require increased time to penetrate the parenchyma,
resulting in their accumulation in extraparenchymal spaces. T
cells also appear to control parasite migration in terms of their
restriction to the CNS during infection. Thus, normal mice that are
i.c. infected with M. corti exhibit an infection confined to
the CNS, whereas TCR-/--infected mice often
show a disseminated infection with encystment of the liver. The
mechanism associated with this remains unclear, although it is possible
that the tremendous accumulation of parasites in the extraparenchymal
areas of mutant mice may physically force the organisms out of
the CNS.
In human NCC, active parenchymal infection is the most common form of
the disease (2, 34, 35). However, it is thought that
symptoms and associated diagnosis occur late in the disease. Parallel
to the human findings, in wild-type mice parasites progressively
penetrate brain parenchyma and the disease worsens as the immune
response develops. As mice often present large number of parasites in
the brain, the magnitude of the disease appears to be a combined effect
of both the presence of parasites in ventricles and brain parenchyma
and the associated inflammatory response. Although T
cell-deficient mice presented the vast majority of parasites in
subarachnoid spaces, they exhibited reduced CNS manifestations and
survive longer. These observations further suggest that the degree of
the host immune response highly correlates with the severity of the
disease.
T cell oligoclonal expansion has been demonstrated in vivo to a
myriad of challenges, including bacterial (36, 37, 38), viral
(19, 39, 40, 41), and parasitic diseases (16, 42, 43). In addition, T cells have been identified in the
brain of NCC patients (Alvarez and Teale, in preparation). T
cells have also been associated with malignancies (39, 44)
and autoimmune diseases (45). In the brain, T cells
have been identified at the margins of chronic multiple sclerosis
plaques, and they appeared to become the prevalent type of T cell
(46). Similarly, T cells were found in the CNS
during EAE (47). Although the role of T cells in
the pathogenesis of EAE appears controversial (48, 49, 50, 51),
studies of T cell-depleted mice suggest that T cells
contribute in the pathogenesis of EAE by regulating the influx of
inflammatory cells and augmenting the proinflammatory cytokine profile
in the brain (50, 51). T lymphocytes bearing the
TCR were also found in the CSF of patients with AIDS-dementia complex
(52) and in CSF of individuals with subacute esclerosis
panencephalitis due to an immune response against measle virus in the
brain (14, 16). The exact contribution of T cells
during such inflammatory disorders involving the CNS remains to be
defined.
The murine NCC model is the first one that describes a predominant
T cell response induced during an infectious state. T
lymphocytes participate as immunoregulatory cells promoting the
development of a type 1 inflammatory response, and appear instrumental
in the immunopathogenesis of murine NCC. T cells amplify the CNS
immune response most likely by indirect activation of several
populations of immune cells (i.e., macrophages, dendritic cells) and
resident brain cells (i.e., microglia, astrocytes). Activated cells
produce diverse cytokines, chemokines, and other soluble mediators that
enhance the inflammatory response and result in further tissue damage.
In addition, the predominant type 1 inflammatory response
down-regulates the type 2 Ab response that usually develops in response
to helminth parasites such as M. corti. Future studies in
this CNS model will be valuable to further understand the effector
functions of T cells and their role in the human disease.
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Footnotes |
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2 Abbreviations used in this paper: NCC, neurocysticercosis; CSF, cerebrospinal fluid; EAE, experimental allergic encephalomyelitis; i.c., intracranial; IHC, immunohistochemistry; p.i., postinfection.
3 Cardona, A., J. Portis, and J. Teale. 2002. / T cells provide a predominant source of IL-12 promoting an inflammatory response in the brain. Submitted for publication.
4 Cardona A, P. Gonzales, and J. Teale. 2002. CC chemokines mediate leukocyte trafficking into the central nervous system during murine neurocycysticercosis: role of T cells in the amplification of the host immune response. Submitted for publication.
Received for publication May 9, 2002. Accepted for publication July 5, 2002.
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