| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Pathology, University of Cambridge, Cambridge, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
During schistosome infection of mice there are dynamic changes in the
prevailing type 1 (IL-2- and IFN-
-secreting cells) or type 2 (IL-4-,
IL-5-, IL-10-, and IL-13-secreting cells) cytokine response. During the
first 4–5 wk of a murine infection there is a type 1 cytokine profile
(6, 7). In this early stage of infection the type 1 response is
reflected by an increase in the numbers of IFN--secreting CD4 (Th1)
and CD8 (Tc1) cells in the spleens and lymph nodes of infected mice
(8). Coincident with patency of the parasite and the commencement of
egg-laying (4–5 wk of infection), type 2 cytokine responses supersede
the early type 1 responses (7). The predominance of type 2 responses by
7–8 wk of infection is associated with a down-regulation of type 1
cytokine responses (7) that is reflected in a loss of Th1 and Tc1 cells
(8, 9). It is known that the egg is the major cause of the development
of a type 2 cytokine response during infection. The potent stimulation
of type 2 cytokines by eggs is evident as cells recovered from naive
mice injected with isolated schistosome eggs secrete type 2 cytokines
(10). The formation of the egg granuloma is a CD4+ T
cell-mediated process (11). The cells that constitute the granuloma
comprise both type 1 and type 2 cytokine-secreting cells (12),
with the cytokine responses within the granuloma controlled by
regulatory cytokines, including IL-10 and TGF-ß (13). Although type 1
and type 2 cytokines are associated with the granuloma, a recent study
in STAT6-deficient mice has illustrated the essential role of type 2
cytokines in the formation of the egg granuloma (14).
As egg/egg Ag-stimulated immune responses are central to the formation of the granuloma we have investigated the effect of eliminating the ability of mice to respond to eggs during infection. Egg Ag-specific unresponsiveness was induced in mice by treatment with the immunosuppressive drug cyclophosphamide (Cy).3 Egg-tolerized mice were infected, and the outcome of infection was examined. Our findings in this model reveal that following schistosome infection the absence of cellular and humoral responses stimulated by eggs, with intact responses to the worms, resulted in a high mortality of egg-tolerized mice. This pathology was associated with type 1 cytokine-dominated responses and diminished type 2 cytokines. These studies show that egg-stimulated type 2 cytokine responses during schistosome infection of mice may regulate proinflammatory type 1 cytokine responses elicited by the other stages of the parasite life cycle.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A Puerto Rican strain of S. mansoni was used in all experiments. The parasite was maintained in laboratory passage in Biomphalaria glabrata snails and outbred Tyler’s Original mice. Female CBA/Ca mice, obtained from Harlan (Bicester, U.K.), were used for all experiments. Mice were maintained under specific pathogen-free conditions. Isolation of schistosome eggs from the livers of infected mice and the preparation of soluble egg Ag (SEA) and adult worm Ag (AW) were previously described (8). OVA was obtained commercially (Sigma, Dorset, U.K.)
Tolerization protocol
Thymectomy was performed on adult mice the day before egg injection. Mice were injected i.p. with 10,000 S. mansoni eggs. The day after egg injection Cy (Sigma) was administered i.p. (200 mg/kg) in divided doses for 4 consecutive days. Four weeks after treatment mice were used in experiments. All animals were placed on antibiotic-supplemented water.
Parasitological techniques
In preliminary studies egg-tolerized mice that were exposed to 120 S. mansoni cercariae had high mortalities. Therefore, the data presented in this paper are from mice that were percutaneously infected with 60 S. mansoni cercariae. Infection and portal perfusion were performed as described previously (15). The liver and intestine were removed and used for tissue egg counts. Tissues were digested in 4% KOH as previously described (16). Fecal samples were collected on the day before termination, and eggs were counted (16). In accordance with our U.K. Home Office regulations, any infected mice that became morbid were humanely killed.
Pathological measurements
To quantify hepatocyte damage, plasma samples were collected from mice during infection or on the day of termination for transaminase assay. Glutamic oxalacetic transaminase (GOT) levels were determined using a commercial kit (Sigma); GOT levels are expressed as Sigma Frankel units per milliliter. The two ventral median lobes of the liver were fixed in formol-saline and embedded in wax, and 5-µm sections were cut. Liver sections were stained with hematoxylin and eosin for granuloma diameter measurement and hepatotoxicity scoring or were stained with Martius Scarlet Blue for examination of fibrosis in the liver. Tissue collagen in the liver was quantified by differential staining of sections (three per mouse) on slides (17). Liver sections were deparaffinized and incubated with a saturated solution of picric acid in distilled water containing 0.1% Fast Green FCF (which stains noncollagenous proteins) and 0.1% Sirius Red F3B (which stains collagen). After 2-h incubation in the dark at room temperature, the slides were rinsed in distilled water until the elution fluid was completely free of color. Each slide was covered in 1 ml of 0.1 N NaOH in absolute methanol (1:1, v/v) for a few seconds until all the color was eluted from the section. The fluid on the sections was carefully transferred to cuvettes, and the absorbance was read at 605 nm (maximal absorbance of Fast Green) and 540 nm (maximal absorbance of Sirius red) on a spectrophotometer. The amount of collagen, expressed as micrograms collagen per milligrams of protein, was calculated using the formula described previously (17). For each mouse the diameters of 21 individual egg granulomas were measured, and livers from 10–15 mice were examined per group (minimum of 210 individual granulomas measured per group). The degree of microvesicular damage to hepatocytes of each mouse was quantified using a previously described arbitrary scale, from 0 (no damaged hepatocytes) to 3 (every hepatocyte in the liver showing evidence of S. mansoni egg-induced microvesicular damage) (18). For studies on the gut, 1-cm sections were fixed in formal-saline for cross-sectional studies, or the entire gut was processed as a Swiss roll. Intestinal sections were hematoxylin and eosin stained, and the localization of the egg and the size of the granuloma were examined. Collagen in the intestine was determined as described above.
Liver and gut pathological measurements were performed in a double-blind fashion. Pathological parameters were measured in at least three separate experiments (with 10–15 mice/group), with consistent results obtained between experiments.
Cell isolation and culture
Spleens, mesenteric lymph nodes (MLN), or popliteal lymph nodes
(PLN) were aseptically removed from age- and sex-matched mice. Two or
three spleens or MLN were pooled, and single cell suspensions were
obtained by passing cells through 70-µm cell strainers (Falcon,
Becton Dickinson Labware, Lincoln Park, NJ). The RBC were
lysed in Tris-ammonium chloride solution. RPMI 1640 (Sigma)
supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin (Life
Technologies, Paisley, Scotland) was used to wash cells. Cells were
stained with trypan blue, and viable cells were counted. The cell
concentrations were adjusted to 5 x 106/ml in culture
medium (RPMI 1640-Dutch Modification; supplemented with 10% FCS
(Sigma), penicillin-streptomycin (50 U/ml to 50 µg/ml), 2-ME (50
µM), and glutamine (2 mM; Life Technologies)). A final cell
concentration of 5 x 106 cells/well was added to a
24-well microtiter plate (Flow, McLean, VA). Cells were stimulated with
10 µg/ml SEA, AW, or anti-CD3 (25 µg/ml; PharMingen, San Diego,
CA). Supernatants were collected after 24 and 72 h of culture and
were stored at -20°C. TNF-
assays were performed immediately on
supernatants collected from 24-h cultures.
For proliferation assays cells were plated (5 x 105/well) on 96-well microplates. Triplicate wells were stimulated with different quantities of Ag (SEA, AW, or OVA) and cultured for 96 h. One microcurie of [3H]thymidine (Amersham, Aylesbury, U.K.) was added for the last 18 h of culture. [3H]thymidine incorporation was monitored and expressed as counts per minute.
Granuloma cells were isolated using methods modified from previously described protocols (19, 20). Briefly, infected mice were portally perfused with cold RPMI 1640 with heparin added. The entire liver was removed and homogenized using a blender (MSE Scientific, Sussex, U.K.). Intact granulomas were isolated by sedimentation (300 x g) and three washes in RPMI 1640. Granulomas were disrupted by incubation in RPMI 1640 with 1500 U/ml collagenase (type IV from Clostridium histolyticum; Sigma) for 35 min at 37°C with constant stirring. After incubation the solution was passed through a 5-ml syringe to disperse the digested granulomas. The granuloma suspension was sieved through 70-µm pore size cell strainers (Falcon, Becton Dickinson, Franklin Lakes, NJ) to remove nondispersed fragments. Granuloma cells were collected after repeated centrifugation and washing in RPMI 1640. Cell viability was determined to be >95% by trypan blue staining and flow cytometry after propidium iodide staining. Granuloma cells were cell phenotyped and cultured with anti-CD3 for 8 h (intracellular cytokine analysis) or for 24 h for cytokine assays on supernatants.
Cytokine ELISA
The levels of IL-2, IFN-, IL-4, IL-5, IL-10, IL-12, TGF-ß1,
and TNF- in cell culture supernatants or serum were quantified using
ELISA. For the assay of IL-2, IFN-, IL-4, IL-5, and IL-10,
anti-cytokine monoclonals and recombinant cytokine standards were
purchased from PharMingen (San Diego, CA) or Genzyme (Kent, U.K.).
Immulon II plates (Dynatech, Chantilly, VA) were coated with 50
µl/well of capture anti-cytokine monoclonal (1–3 µg/ml) in 0.1
M Na2HPO4 (pH 9.0) overnight at 4°C. PBS-T
(PBS with 0.05% Tween-20) was used to wash plates, and they were
blocked for 1 h in 1% BSA in PBS-T. Serial dilutions of
recombinant cytokine or cell supernatants were added (50 µl/well) for
2 h at room temperature. Biotinylated anti-cytokine detection
mAb (0.5–2 µg/ml) were added for 2 h at room temperature. ABTS
(2,2'-azino-bis(3-ethybenz-thiazoline-6-sulfonic acid)) was
used as substrate, and plates were read at 405 nm. The concentrations
of IL-2 (picograms per milliliter), IL-4 (units per milliliter), IL-5
(nanograms per milliliter), IL-10 (units per milliliter), and IFN-
(nanograms per milliliter) were interpolated from the appropriate
recombinant cytokine standard curve. TNF- was quantified using a
commercial ELISA kit according to the manufacturer’s instructions
(Genzyme, Cambridge, MA). Total TGF-ß1 (acidified samples) was
measured with a commercial assay according to the manufacturer’s
instructions (Promega, Southhampton, U.K.). IL-12 was measured with
paired mAb and recombinant cytokine that were gifts from Cambridge
Bioscience (Cambridge, U.K.).
Flow cytometry and intracellular cytokine detection
Intracellular cytokine staining was used to determine the
frequency of IL-4- or IFN--producing CD4+ and
CD8+ T cells as previously described (8). All reagents for
intracellular cytokine staining were purchased from Caltag (Burlingame,
CA). Cells were cultured at 106/ml in 24-well plates
(Falcon) for 8 h in the presence of medium alone or with
plate-bound anti-CD3 (25 µg/ml; PharMingen). Brefeldin A (5
µg/ml; Sigma) was added for the last 3 h of culture. Cells
(106/well) were transferred to 96-well U-bottomed
microtiter plates (Dynatech). Cells were blocked for 10 min on ice in
50% FCS in FACS buffer (PBS/1% FCS). TRI-Color-conjugated
anti-mouse CD4 or CD8 was added for 45 min on ice to phenotype
cells. After washing, cells were fixed in fixation medium (Caltag).
Permeabilization of cells occurred in the presence of FITC-conjugated
anti-mouse IFN- and phycoerythrin-conjugated anti-mouse
IL-4. After washing cells were stored overnight at 4°C in FACS
fixative buffer (FACS buffer with 1% paraformaldehyde). Flow cytometry
was performed on a FACScan with LYSIS II software (Becton Dickinson
Immunocytometry Systems, San Jose, CA). In all experiments unstained
cells and cells stained separately with each flurochrome were included
to optimize compensation settings. The addition of excess recombinant
cytokines or unlabeled anti-IL-4 or IFN- mAb was used to confirm
that the cytokine signals detected were specific. Lymphocytes were
gated on their forward and side scatter characteristics. TRI-Color
(FL3)-stained CD4 or CD8 lymphocytes were gated, and 15,000 events were
analyzed per sample. The percentage of CD4+ or
CD8+ T cells containing intracytoplasmic IL-4 or IFN- in
appropriate quadrants was determined. Three replicates of stained cells
were FACS acquired/analyzed separately in each experiment, and the mean
positive cells of the triplicate determinations was calculated.
Results are presented as the mean percentage of positive cells obtained
from at least three different experiments. Cells from the spleen or
granulomas of individual mice were phenotyped with flurochrome-labeled
anti-CD3, anti-CD4, anti-CD8, and B220.
ELISA for Ag-specific Ab responses
Sera were collected from mice before infection and on the day of termination. IgG, IgG1, IgG2a, and IgE isotype responses to AW, SEA, and OVA were evaluated using an ELISA. Optimum conditions for ELISA were obtained by checkerboard titrations with pooled positive (from 20-wk-infected mice) and negative (uninfected mice) serum. Microtiter plates were coated overnight with Ag. After three washes in PBS-T, plates were blocked with 1% BSA. Individual mouse serum (1/200 for IgG, IgG1, and IgG2a and 1/25 for IgE) was added in triplicate wells. IgG, IgG1, and IgG2a were detected with peroxidase-conjugated anti-mouse isotype Ab (Sigma or Zymed (San Francisco, CA)). For detection of IgE, plates were incubated with biotinylated anti-mouse IgE followed by streptavidin-peroxidase. Three separate experiments were analyzed; in each experiment 10–15 mice were used in each group.
Statistical analysis
Statistical differences between groups were determined using Student’s t test. A p value of <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Administration of Cy with Ag induces a state of Ag-specific
tolerance (21). We used Cy treatment to induce tolerance of mice to
schistosome eggs. Mice were injected with schistosome eggs and treated
with Cy; control groups included mice injected with eggs alone or mice
treated with Cy alone. Four weeks after treatment mice were injected
with 5000 eggs into the hind footpads, and 14 days later the draining
PLN were removed. Cells from the PLN were restimulated in vitro with
varying quantities of SEA, and proliferation responses were determined
by [3H]thymidine incorporation. The Cy(+) egg-injected
mice had substantially impaired proliferation responses to SEA compared
with the responsiveness of the other groups (Fig. 1
A). To test whether the responses to all Ag
were impaired in the Cy(+) egg-treated mice, OVA was injected into the
footpad, and proliferation responses to OVA were assayed. The Cy(+)
egg-treated group had similar proliferation responses to OVA as control
groups (Fig. 1B). As humoral responses are also modified by
Cy-induced tolerance, we measured Ab responses to SEA or OVA. Cy(+)
egg-treated mice had impaired Ab responses to SEA, but mounted normal
Ab responses to OVA (not shown). The treatment of mice with Cy after
injection with S. mansoni eggs induces a state of Ag
specific tolerance to parasite egg Ag.
|
Effects of egg tolerization on schistosome infection
During initial studies egg-tolerized animals were infected with
120 S. mansoni cercariae. By day 50 after infection there
were high mortalities (>90%) of the egg-tolerized mice, with no
deaths by this stage of infection in any control animals. On the basis
of these results additional experiments were performed using a lighter
infection, with mice exposed to 60 cercariae. In three separate
experiments following exposure of mice to 60 cercariae there was high
mortality (>80%) of egg-tolerized mice by the eighth week of
schistosome infection, whereas <5% of mice in thymectomized,
Cy-treated, egg-injected or combined thymectomy and Cy-treated control
groups had died. As shown in Fig. 2, mice were dying
from day 45 after infection, i.e., within approximately 14 days of the
onset of egg laying; by the 51st day after infection >50% of the
egg-tolerized mice had died, with these deaths occurring within a
period of 6 days (days 45–51). Egg-tolerized mice with lighter
infections (exposure to 25 cercariae) also suffered high mortalities
(>70%) by the 8–9th week of infection. In all cases, regardless of
the level of parasite infection, death of egg-tolerized mice occurred
during the acute stages of infection. In all experiments low
mortalities (<5%) were observed in all control (thymectomized,
Cy-treated, egg-injected, or combined thymectomy and Cy-treated)
groups. The early mortality of egg-tolerized mice during infection
prevented studies during the chronic stage of infection. We therefore
focused on pathology and immune responses in egg-tolerized mice during
the early acute (7th week) stages of infection.
|
). These data indicated that the death of egg-tolerized
mice was not due to any direct effect of treatment on the parasite.
Egg-tolerized mice had dramatically reduced egg excretion, with >90%
fewer eggs being excreted in the feces of tolerized mice compared with
control animals (Table I). The inability of egg-tolerized animals to
excrete eggs in the feces was partially reflected in marginal higher
levels of eggs present in the intestine of these mice compared with
those in control infected mice (Table I).
|
A). The sizes of granulomas surrounding eggs
in the livers of tolerized mice were significantly smaller
(p < 0.001) than those of granulomas in
control infected animals (Table II and Fig. 3, B and C). Similarly, we have observed that
pulmonary granuloma after i.v. injections of eggs are diminished in
size in egg-tolerized animals (not shown). We also measured collagen
within the liver, as fibrosis is a sequel to egg granuloma formation
during schistosomiasis. Egg-tolerized mice had a significant reduction
in collagen deposition in liver tissue (Table II). Martius Scarlet Blue
staining visually demonstrated the dramatically smaller granulomas
surrounding the schistosome egg and also the reduced amount of
collagenous material (blue stain) in the liver of egg-tolerized animals
compared with those in control animals (Fig. 3, B and
C). Sections of liver from egg-tolerized mice showed
extensive hepatocyte damage; this cell damage (e.g., loss of
cytoplasmic staining in hepatocytes) was external to the granuloma and
was dispersed in the liver parenchyma (Fig. 3E). Higher
magnification revealed ballooning degeneration and microvesicular
steatosis of hepatocytes in egg-tolerized animals (Fig. 3G).
There was limited hepatocyte damage in infected control animals (Fig. 3, D and F). Using a previously defined arbitrary
scale to quantify hepatocyte damage in schistosome-infected mice (18),
double-blind scoring of liver sections confirmed the extensive
hepatocyte damage in the egg-tolerized mice (Table II). We also
measured the plasma levels of GOT, as plasma transaminases are markers
for hepatocyte damage. Infected egg-tolerized mice, had significantly
elevated levels of GOT (p < 0.001) compared
with control infected animals (Table II). These effects on the liver
were not directly attributable to an increase in the numbers of
schistosome eggs in the livers of egg-tolerized mice, as they had
comparable numbers of eggs in the liver as control animals that had
limited liver damage (Table I
). When we studied the livers of
uninfected animals there was no difference (liver collagen, hepatocyte
integrity, or plasma GOT levels) between egg-tolerized or control mice.
Collectively, these data demonstrated that following schistosome
infection the livers of egg-tolerized mice had severe pathology that is
associated with extensive hepatotoxicity.
|
|
), we also examined the guts of these
animals. Tissue digests of liver or guts from infected mice showed that
there was no significant difference in the relative distribution of
eggs in the liver or gut between egg-tolerized and control groups
(Table I); this indicates that the failure of egg-tolerized mice to
excrete eggs is not due to aberrant egg deposition by the parasite.
Histologically, the intestines of infected normal mice were
characterized by the appearance of granulomas surrounding eggs in the
submucosa, with eggs also detected within granulomas in the lamina
propria and along the villi, and with eggs in various stages of
extravasation to the lumen (Fig. 3H). In contrast, in all
sections of intestines from egg-tolerized mice, eggs were predominately
detected in the submucosa, with few eggs detected in the lamina propria
or ascending the villi. No or limited granulomatous responses were
apparent in the vicinity of eggs in the intestine of egg-tolerized mice
(Fig. 3I). Similar to the hepatic granuloma, there were
fewer eosinophils detected in the vicinity of the egg in the intestine.
An additional observation was that there was significantly more
(p < 0.001) collagen in the intestines of
infected egg-tolerized mice (12.6 ± 1.28 µg/mg; mean of 10
infected mice) compared with that in control infected mice (10.6
± 0.8 µg/mg; mean of nine mice). However, the levels of collagen in
uninfected egg-tolerized mice were also significantly higher
(p < 0.01) than those in uninfected control
animals (not shown). This increase in intestinal collagen in
egg-tolerized mice is due to the Cy treatment, as mice treated with Cy
also had increased intestinal collagen. However, mice that were only
treated with Cy had normal egg excretion during schistosome infection. Cellular and humoral responses in schistosome-infected egg-tolerized mice
Spleen cells from thymectomized, Cy-treated, egg-injected,
combined thymectomized and Cy-treated, or Cy-treated, thymectomized,
and egg-injected mice were analyzed by flow cytometry to ascertain
whether the treatment protocol affected CD4+,
CD8+, and B220+ cell populations. The combined
thymectomy and Cy treatment, with or without concurrent egg injection,
induced a transient lymphopenia in mice, with a 20–50% reduction in
CD3+ or B220+ cells in the spleen detected
24 h after the last Cy treatment. By the day of infection (4 wk
after Cy treatment) spleen lymphocyte numbers (CD3+,
CD4+, CD8+, B220+) had returned to
pretreatment levels. This recovery of lymphocyte populations after
combined Cy treatment and thymectomy of adult mice has been reported
previously (24). Spleens and MLNs were removed from mice during the
seventh week of schistosome infection. Spleen cells from infected
egg-tolerized mice were restimulated in vitro with AW and SEA to
determine whether proliferation responses to parasite Ag were impaired
after infection. Cells from egg-tolerized mice had impaired
proliferation responses when restimulated with a range of
concentrations of SEA, whereas control infected animals were responsive
to SEA (Fig. 1C). However, consistent with the tolerance
being egg Ag specific, egg-tolerized mice had unimpaired proliferation
responses to worm Ag, with AW responses comparable to those in control
animals (Fig. 1D).
To evaluate what cytokine responses were elicited during schistosome
infection, cells from infected mice were cultured in vitro in the
presence of parasite Ag (AW or SEA) or were stimulated with
anti-CD3. ELISA of the supernatants from cultures demonstrated that
when stimulated with anti-CD3, cells from control infected animals
produced predominately type 2 cytokines (Fig. 4). In
contrast, anti-CD3-stimulated cells from egg-tolerized mice
produced significantly more type 1 cytokine (IL-2, IFN-, and
TNF-; Fig. 4, A–C) and significantly less type 2
cytokine (IL-4, IL-5, and IL-10; Fig. 4, D–F) than control
animals. SEA, a known stimulator of type 2 cytokines during schistosome
infection, elicited the secretion of IL-4, IL-5, and IL-10 in control
animals, but, consistent with the cell proliferation data, this Ag
preparation stimulated limited cytokine secretion by cells from
egg-tolerized mice. Restimulation of cells from infected egg-tolerized
mice with AW caused the abundant production of IFN- (four- to
fivefold more produced than in cells from control infected animals),
IL-2 (two- to threefold higher levels), and TNF- (two- to threefold
greater levels); with AW not stimulating the secretion of type 2
cytokines. Similar cellular responses were observed in MLN cells. These
data reveal that by the seventh week of schistosome infection
egg-tolerized mice have a type 1 cytokine-dominated response that is
also associated with diminished type 2 cytokines, with AW stimulating
type 1 cytokine secretion by cells from egg-tolerized mice. In the
context of the type 1/type 2 cytokine dichotomy the cytokine profile of
infected egg-tolerized mice is almost the complete opposite of that of
the type 2 cytokine-dominated response observed in normal infected
mice.
|
). Egg-tolerized mice had similar
total IgG responses to AW as control infected animals (Fig. 5).
However, egg-tolerized animals had marginally increased IgG2a responses
and a significantly diminished production of IgG1 and IgE isotypes to
AW (p < 0.001). The skewing in anti-worm
Ab isotypes to an IgG2a-dominated response with absence of IgG1 and IgE
in egg-tolerized animals may reflect the worm Ag-driven type 1 cytokine
environment (Fig. 4) of these animals.
|
Characteristically, during the course of schistosome infection of
mice there is a gradual switch from a predominant type 1 cytokine
response during the first 4 wk of infection to a type 2
cytokine-dominated response after egg laying (6, 8). However, as we
have shown above, egg-tolerized mice have a type 1 cytokine-dominated
response with diminished type 2 cytokines by the seventh week of
infection. Thus, we investigated whether this type 1 cytokine bias
occurred earlier in infection. Spleen cells were removed from infected
mice on day 28 (before egg laying; when type 1 responses prevail) and
day 47 after infection (the post-egg-laying phase when egg-driven type
2 responses predominate). Cells were restimulated with anti-CD3.
IFN- and IL-4 in cell supernatants were assayed as respective
representative type 1 and type 2 cytokines; we also measured IL-10, as
this cytokine has been shown to regulate type 1 responses during acute
schistosome infection (25, 26). CD3-stimulated cells from normal
infected mice displayed IFN- secretion and limited IL-4 secretion
when examined 28 days after infection; later during infection this
response was superseded by a type 2 dominated response, with reduced
IFN- production and marked enhanced secretion of IL-4 (Fig. 6, A and B). Egg-tolerized mice
had similar IFN- responses as control animals by day 28 after
infection, but there was consistently marginally more production of
this cytokine by cells from tolerized animals at this stage of
infection (Fig. 6A). By day 47 of infection the cytokine
responses of cells from egg-tolerized mice were strikingly different
from responses of control animals; there was an increase in the
secretion of IFN- and, conversely, no elevation in IL-4 secretion
(Fig. 6, A and B). In normal animals the increase
in IL-10 secretion was coincident with the reduction in IFN- release
by cells (Fig. 6C). In egg-tolerized mice when we examined
IL-10 secretion we observed limited production of this cytokine during
infection (Figs. 4 and 6C). The absence of IL-10 and IL-4 in
acute infection may be partially responsible for the inability of
tolerized mice to down-regulate type 1 cytokine responses that are
induced by larval or worm Ag early during schistosome infection.
|
, D and E). In
contrast to the marked reduction by day 47 postinfection in the
frequency of Tc1 or Th1 cells observed in normal mice, there was a
limited decline in these cell populations in egg-tolerized mice.
Egg-tolerized mice also did not have the expansion in the frequencies
of Th2 cells that occurred in control animals later during infection
(Fig. 6F). A type 1 cytokine environment prevails within the liver granuloma of egg-tolerized mice
The cells that constitute the liver granuloma have been used
previously to analyze the cytokine responses that intimately regulate
formation of the egg granuloma (13, 20). To address the question of
whether the generalized type 1 cytokine responses in egg-tolerized
animals was reflected in granuloma cell populations, we isolated
granuloma cells from the livers of mice from 6–7 wk after infection.
Initial attempts using standard protocols (19, 20) to isolate cells
from liver granulomas of egg-tolerized mice were confounded by the high
levels (>50%) of propidium iodide-stained (dead) cells detected by
flow cytometry, compared with <5% stained cells that were recovered
from granulomas of normal mice. By reducing the amount of collagenase
used to disrupt the intact granuloma, the levels of cell damage was
reduced <5% in egg-tolerized mice, but this amount of enzyme failed
to adequately separate granuloma cells from the livers of normal
infected mice (the requirement for more collagenase treatment to
isolate cells from the granulomas of infected mice compared with
egg-tolerized mice is probably due to the greater amount of collagen in
livers of normal mice compared with that in egg-tolerized mice; Table II). Therefore, by extending the collagenase digestion incubation time
to 35 min, <5% damaged cells (propidium iodide) were recovered from
liver granulomas from mice. Consistently, three- to sixfold fewer
granuloma cells were recovered from the livers of individual
egg-tolerized mice compared with cell recovery from normal infected
mice; this reduction in granuloma cell recovery may be a reflection of
the smaller sized granulomas in these animals (Table II).
ELISA detection of IL-4 and IFN- in the supernatants from 24-h
cultures of anti-CD3-stimulated granuloma cells showed that there
was a greater propensity for granuloma cells from egg-tolerized animals
to secrete IFN- and, conversely, a diminished capacity to produce
IL-4 compared with secretion of these cytokines by granuloma cells from
normal infected mice (Fig. 7, A and
B). Using intracellular cytokine staining it was shown that
granuloma cells from normal infected mice had a mixed composition of
IFN-- and IL-4-stained CD4 and CD8 cells; within CD8 cells Tc1 were
more abundant, and conversely with respect to CD4+ cells
there was approximately a 2:1 ratio of Th2 cells to Th1 (Fig. 7, C and D). There were negligible frequencies
(<0.01%) of IL-4-stained CD4+ or CD8+ cells
within granuloma cells recovered from egg-tolerized livers (Fig. 7, E and F). In contrast, there was a marked
increase in the frequencies of Tc1 (>4% positive) and Th1 (>5%
positive) cells within granuloma cell populations from egg-tolerized
mice. The detection by flow cytometry of predominately
IFN--secreting CD4+ or CD8+ cells in
granulomas of egg-tolerized animals suggests that in these animals
there are increases in the frequencies of IFN- production by T cells
within the egg granuloma and a reciprocal reduction in IL-4-secreting
cells.
|
and IL-12 by granuloma cells from egg-tolerized
mice was considerably higher than secretion of these cytokines by
granuloma cells from normal infected animals (Table III). Consistent with the granuloma of egg-tolerized
mice being type 1 cytokine dominated, there was reduced IL-10 and IL-4
secretion by granuloma cells from these mice compared with that by
cells from normal infected mice (Fig. 7 and Table III). The secretion
of TGF-ß1, a regulatory cytokine with respect to IFN-, by
granuloma cells from egg-tolerized mice was sevenfold less than the
secretion of this cytokine by granuloma cells from normal mice (Table III). The relatively reduced secretion of IL-10 and TGF-ß1 by
granuloma cells from egg-tolerized mice and the increased IL-12 levels
may be driving the increased secretion of IFN- and TNF- within
the granuloma.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In earlier studies, McCurley and colleagues (28) attempted to induce unresponsiveness to schistosome eggs using various protocols to induce tolerance, including intrathymic injection of SEA, oral administration of eggs or SEA, and high and low dose Ag injection. After i.v. administration of egg Ag there was an indication that following high dose injection a state of tolerance was induced, i.e., mice injected twice with 1 mg of SEA had smaller pulmonary granulomas than control PBS-injected animals. All other tolerance protocols attempted failed to cause unresponsiveness (28). Similarly, mice treated with SEA and Cy have been shown to have smaller pulmonary granulomas (29). These two studies used soluble egg Ag to induce unresponsiveness, whereas in this study we used whole eggs. We induced partial unresponsiveness to SEA using Cy treatment and also observed smaller pulmonary granulomas. However, following schistosome infection mice that are rendered unresponsive to SEA have normal hepatic granulomas (not shown). That responses that participate in the formation of the schistosome egg granulomas in the lungs and the liver may differ has also been observed previously in normal (30) and IL-4-deficient (31) mice.
Following schistosome infection of STAT6-deficient mice, pathology
similar to that described here occurs (smaller granuloma and reduced
hepatic collagen) with cytokine responses comparable to the data
obtained here with egg-tolerized mice, i.e., a generalized skewing to a
type 1 cytokine profile (14). Data from studies on IL-4-deficient mice,
which may be influenced by experimental conditions and the genetic
background of the animal, have also shown that there is a modified
pulmonary (naive mice injected with eggs i.v.) (31) and hepatic
granulomas (eggs deposited in situ) (32) in these animals. Excessive
hepatic collagen deposition and the formation of fibrosis are causes of
pathology during schistosome infection. In egg-tolerized mice there was
a significant reduction in the amount of hepatic collagen. The elevated
IFN- secretion by cells recovered from within the hepatic granuloma
of egg-tolerized mice may be relevant, as IFN- can regulate collagen
formation (33). Indeed, administration of recombinant IFN- to
schistosome-infected mice has been shown to reduce collagen deposition
(34, 35). The relatively lower amounts of TGF-ß1 in the granuloma
cells of egg-tolerized mice will also have implications for liver
fibrosis due to the known fibrogenic properties of this cytokine (36).
In murine schistosomiasis TGF-ß1 has been shown to be elevated within
the liver, and the cytokine is associated with fibrosis (37).
Additionally, in TGF-ß1 transgenic mice liver collagen synthesis is
increased during schistosome infection (38). Collectively, our data
suggest that the dominance of a type 1 cytokine phenotype concurrently
associated with the presence of a diminished type 2 cytokine response
impairs the development of schistosome egg granulomas and reduces
collagen (fibrosis) formation in acutely infected mice.
We have recently shown that during the development of a type 2
cytokine-dominated response in schistosome infection there is a
reduction in the relative frequency of IFN--producing
CD8+ T cells (Tc1 cells) and an expansion of IL-4-producing
CD4+ T cells (Th2 cells) (8). In this study egg-tolerized
mice had a type 1 cytokine profile by day 47 after infection, which is
in marked contrast to the predominance of a type 2 cytokine response at
this time of infection in normal mice (Fig. 4). The type 1
cytokine-dominated responses in egg-tolerized mice were in part due to
a failure to reduce the number of Tc1 cells (Fig. 6). This inability to
down-regulate these cells may be due to the absence of IL-10. Recently,
it has been shown that during this acute stage of infection IL-10 may
mediate activation-induced cell death of certain cell populations (9).
The crucial role of IL-10 in murine schistosome infection has been
shown previously. Thus, treatment of schistosome-infected mice with
exogenous IL-10 has been shown to directly regulate cellular responses
and reduce immunopathological damage (39, 40). Additionally, Wynn and
colleagues (26) have demonstrated that in the absence of IL-10
(IL-10-deficient mice) there is an elevated type 1 cytokine profile
with impaired granuloma formation and increased pathology during acute
schistosome infection. In double IL-10- and IL-4-deficient or IL-10-
and IL-12-deficient animals the central regulatory role for IL-10 in
responses to schistosome eggs has been further delineated (41).
In various infectious diseases it is becoming increasingly apparent
that the intimate cytokine milieu regulates susceptibility and
pathology during infection (42). In the context of schistosome
infection it is the egg granuloma that tightly regulates local cytokine
production. Recently, it has been shown that IFN- production by
cells within the granuloma is regulated by the presence of IL-4, IL-10,
and TGF-ß in the granuloma microenvironment (13). In this study
granuloma cells from normal infected mice had a mixed production of
type 1 and type 2 cytokines, with the coincident presence of IL-4- and
IFN--secreting T cells as well as cell-derived IL-10 and TGF-ß1
(Table III). In contrast, cells from the liver granulomas of
egg-tolerized mice secreted significantly more IFN-, TNF-, and
IL-12, with reduced IL-4, IL-10, and TGF-ß1. Thus, the cytokine
environs of the granulomas of egg-tolerized mice are type 1 cytokine
dominated, which may be due to the absence of IL-4 and conversely the
presence of elevated IL-12 within the granuloma. The increased
secretion of IL-12 by granuloma cells from egg-tolerized mice is
probably relevant to liver damage, as in other type 1 cytokine-induced
models of severe liver injury a central role for IL-12 has been shown
(43). The schistosome granuloma, via, for example, the known
counter-regulatory role of IL-4 and IFN- on TGF-ß (44), is a
tightly controlled event that is dysfunctional in tolerized mice.
The tolerization method adopted in this study (Cy plus Ag) has
previously been shown to influence B cell function and impair
Ag-specific Ab responses (21, 22). Consistent with an effect on B
cells, infected egg-tolerized mice had significantly reduced levels of
Ab to egg Ag in ELISA. This humoral defect was Ag specific, as
egg-tolerized mice had similar levels of total IgG to worm Ag as
control animals. However, the anti-worm Ab response was type 1
isotype biased, with elevated IgG2a and diminished IgG1 and IgE in
infected tolerized mice relative to those in control animals. As IgG2a
isotype responses are stimulated by IFN- (45, 46, 47), the
IFN--dominated responses throughout schistosome infection of
tolerized mice will have promoted the expression of this isotype.
Conversely, the absence of type 2 cytokines (IL-4 and IL-13) that
regulate class switching to IgG1 and IgE (48) in tolerized mice will
have prevented the expansion of these isotypes during infection.
The major pathology observed in egg-tolerized mice was extensive damage
to the liver. During experimental schistosome infections of nude, SCID,
and T cell-depleted mice there is also extensive hepatotoxicity, which
is characterized by similar cell damage and microvesicular steatosis as
described here (2, 3, 4, 5). Although it is not known whether similar liver
pathology occurs in humans, individuals coinfected with schistosomiasis
and HIV have raised plasma transaminase levels, suggesting elevated
hepatocyte damage in immunocompromised humans (49). In all these cases
of hepatotoxicity there is a defect in anti-egg immune responses
during infection and a quantitative and qualitative difference in the
granulomatous response to the egg. Egg-tolerized mice start to die
within 2 wk of eggs arriving in the liver; thereafter, 50% of mice
succumb to liver damage within 1 wk. The absence of Ab to egg Ag in
tolerized mice is of particular significance with respect to the
hepatotoxicity in these mice. T cell- and CD4+-depleted
mice also have diminished Ab responses to egg Ag, and these mice suffer
severe microvesicular damage during infection; this hepatocyte damage
can be prevented by the transfer of serum from infected mice (18)
(P. G. Fallon, unpublished observations). It is relevant that this
hepatocyte damage occurs in immunologically intact mice once egg laying
starts and eggs are deposited in the liver (
30–35 days after
infection), but in normal mice the hepatocyte damage is transient and
is resolved by emerging Ab and/or type 2 cytokine responses. Thus, the
presence of anti-egg Ab during schistosome infection may have a
crucial role, by neutralizing/removing secreted egg Ag, in ameliorating
hepatocyte damage. A potential protective role for Ab has also been
shown during schistosome infection of B cell-deficient (µMT) mice.
These mice have enlarged granulomas and elevated hepatic collagen with
increased mortality compared with wild-type mice. This pathology was
not associated with a change in the type 1/type 2 cytokine balance
(50). However, it is relevant that in a separate study in a different B
cell-deficient mouse (JH locus deleted) there was a type 1
cytokine-dominated response during acute schistosome infection with
normal granulomatous responses (51). Jankovic and colleagues suggest
that as FcR-deficient mice also have similar liver pathology as
µMT mice the role of Ab in regulating the granulomatous response to
schistosome eggs is via Ab directly stimulating the production of
anti-inflammatory mediators from FcR+ cells (50, 52).
As shown in this study egg-tolerized mice have a dramatically impaired (>90%) capacity to excrete parasite eggs. The schistosome egg excretion process requires eggs that are laid by adult worms in the mesenteric vasculature to transverse the intestinal wall to enter the gut lumen. Egg excretion is an immune-dependent process, with reduced excretion of eggs in T cell-deprived mice (16), SCID mice (5), more recently in B cell-deficient (µMT) mice (50), and CD4+ cell-depleted mice (P. G. Fallon et al., unpublished observations). Data from schistosome-infected humans support the hypothesis that egg excretion is immune dependent, with individuals with reduced CD4+ cells having reduced egg excretion (49). In egg-tolerized mice and CD4+ T cell-depleted animals there is a diminished granulomatous response associated with reduced type 2 cytokines and a quantitative defect in anti-egg Ab production during schistosome infection (this study and our unpublished observations). One mechanism by which type 2 cytokines could be active in facilitating schistosome egg excretion is via IL-5-mediated eosinophil production (53). In this study egg-tolerized mice have limited IL-5 secretion, and although we have not quantified blood eosinophils there were fewer eosinophils within the egg granuloma. The presence of eosinophils in the intestine has been implicated in the passage of eggs through the intestine (54). Thus, the absence of anti-egg immune responses in egg-tolerized mice is clearly a factor in the impaired egg excretion of these animals. However, it is also probable that the elevated gut collagen formation in these animals is relevant. Cy treatment kills dividing cells, and as the gut is a site of rapid cell turnover, there will be some intestinal cell damage and fibrosis in Cy-treated mice. In this study control mice that were thymectomized and Cy treated but not injected with eggs had the same elevated intestinal collagen levels as tolerized mice, but these animals had normal egg excretion. Additional studies are required to elucidate the immune mechanisms that control passage of schistosome eggs through the intestine.
In conclusion, this experimental study demonstrates that rendering mice unresponsive to eggs causes elevated type 1 cytokine responses with diminished type 2 responses during schistosome infection, and these effects are detrimental to the host. The results reported here support observations in a previous study in IL-4-deficient mice suggesting a potential protective role for type 2 cytokines in schistosome infection (55). This study has demonstrated that the normal stimulation of type 2 cytokines by eggs during schistosome infection is essential to regulate the proinflammatory type 1 cytokines that are elicited during early schistosome infection.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Padraic Fallon, Department of Pathology, University of Cambridge, Tennis Court Rd., Cambridge, United Kingdom CB2 1QP. E-mail address:
3 Abbreviations used in this paper: Cy, cyclophosphamide; SEA, soluble egg antigen; AW, adult worm antigen; GOT, glutamic oxalacetic transaminase; MLN, mesenteric lymph nodes; PLN, popliteal lymph nodes.
Received for publication September 3, 1998. Accepted for publication December 22, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature 356:604.[Medline]
(IFN-), IL-4 and IL-10 in schistosome egg granuloma formation: in vivo regulation of Th activity and inflammation. Clin. Exp. Immunol. 98:395.[Medline]
production within the granulomas of murine schistosomiasis in IL-4 deficient and control mice. J. Immunol. 160:4994. secretion. J. Immunol. 149:3621.[Abstract]
-Interferon treatment inhibits collagen deposition in murine schistosomiasis. Hepatology 10:795.[Medline]
. J. Hepatol. 26:894.[Medline]
have opposing effects, while TGF-ß positively regulates its own production. J. Immunol. 160:5719. regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol. 140:1022.[Abstract]
enhances secretion of IgG2a from IgG2a-committed LPS-stimulated murine B-cells: implications for the role of IFN- in class switching. Cell. Immunol. 135:95.[Medline]
R1-deficient mice infected with Schistosoma mansoni mount normal Th2-type responses while displaying enhanced liver pathology. J. Immunol. 59:1868.
mediated cachexia and death during acute schistosomiasis. J. Immunol. 159:777.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. P. Saunders, C. M. Walsh, J. L. Barlow, N. E. Mangan, P. R. Taylor, A. N. J. McKenzie, P. Smith, and P. G. Fallon The C-Type Lectin SIGNR1 Binds Schistosoma mansoni Antigens In Vitro, but SIGNR1-Deficient Mice Have Normal Responses during Schistosome Infection Infect. Immun., January 1, 2009; 77(1): 399 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Swartz, K. D. Dyer, A. W. Cheever, T. Ramalingam, L. Pesnicak, J. B. Domachowske, J. J. Lee, N. A. Lee, P. S. Foster, T. A. Wynn, et al. Schistosoma mansoni infection in eosinophil lineage-ablated mice Blood, October 1, 2006; 108(7): 2420 - 2427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. E. Mangan, N. van Rooijen, A. N. J. McKenzie, and P. G. Fallon Helminth-Modified Pulmonary Immune Response Protects Mice from Allergen-Induced Airway Hyperresponsiveness J. Immunol., January 1, 2006; 176(1): 138 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Stavitsky Regulation of Granulomatous Inflammation in Experimental Models of Schistosomiasis Infect. Immun., January 1, 2004; 72(1): 1 - 12. [Full Text] [PDF]
|
|
|
|
|
|
C. Eboumbou Moukoko, N. El Wali, O. K. Saeed, Q. Mohamed-Ali, J. Gaudart, A. J. Dessein, and C. Chevillard No Evidence for a Major Effect of Tumor Necrosis Factor Alpha Gene Polymorphisms in Periportal Fibrosis Caused by Schistosoma mansoni Infection Infect. Immun., October 1, 2003; 71(10): 5456 - 5460. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Buendia, L. Del Rio, N. Ortega, J. Sanchez, M. C. Gallego, M. R. Caro, J. A. Navarro, F. Cuello, and J. Salinas B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection Infect. Immun., December 1, 2002; 70(12): 6911 - 6918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Saraiva, P. Smith, P. G. Fallon, and A. Alcami Inhibition of Type 1 Cytokine-mediated Inflammation by a Soluble CD30 Homologue Encoded by Ectromelia (Mousepox) Virus J. Exp. Med., September 16, 2002; 196(6): 829 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Fallon, P. Smith, E. J. Richardson, F. J. Jones, H. C. Faulkner, J. Van Snick, J.-C. Renauld, R. K. Grencis, and D. W. Dunne Expression of Interleukin-9 Leads to Th2 Cytokine-Dominated Responses and Fatal Enteropathy in Mice with Chronic Schistosoma mansoni Infections Infect. Immun., October 1, 2000; 68(10): 6005 - 6011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. F. Hoffmann, A. W. Cheever, and T. A. Wynn IL-10 and the Dangers of Immune Polarization: Excessive Type 1 and Type 2 Cytokine Responses Induce Distinct Forms of Lethal Immunopathology in Murine Schistosomiasis J. Immunol., June 15, 2000; 164(12): 6406 - 6416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Fallon, E. J. Richardson, G. J. McKenzie, and A. N. J. McKenzie Schistosome Infection of Transgenic Mice Defines Distinct and Contrasting Pathogenic Roles for IL-4 and IL-13: IL-13 Is a Profibrotic Agent J. Immunol., March 1, 2000; 164(5): 2585 - 2591. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. McKenzie, P. G. Fallon, C. L. Emson, R. K. Grencis, and A. N.J. McKenzie Simultaneous Disruption of Interleukin (IL)-4 and IL-13 Defines Individual Roles in T Helper Cell Type 2-mediated Responses J. Exp. Med., May 17, 1999; 189(10): 1565 - 1572. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
